In vivo study and thermodynamic investigation of two lanthanum complexes, La(dpp)3 and La(XT), for the treatment of bone resorption disorders.

Bone density diseases such as osteoporosis affect a significant number of people worldwide. Lanthanide ions are functional mimics of calcium ions, able to substitute for Ca2+ in the bone mineral component, hydroxyapatite (HAP). Bone undergoes a continuous remodelling cycle and lanthanides can affect this cycle, exerting a positive influence on bone mineral. We have been engaged in efforts to find new lanthanide containing complexes as active agents for treatment of these diseases and have identified two lead compounds, 3-hydroxy-1,2-dimethylpyridin-4(1H)-one (Hdpp) and a phosphinate-EDTA derivative, bis[[bis(carboxymethyl)amino]-methyl]phosphinate (H5XT). In this paper, we report in vivo data for the first time for the two lead compounds. The pharmacokinetics of La(dpp)3 suggest the complex is rapidly cleared from plasma. We demonstrate that La3+ accumulates in the bone following IV dose of either La(dpp)3 or La(XT) and we have investigated the influence of each chelating ligand on the incorporation of La3+ into HAP using ITC and HAP-binding studies.

Binding specificity and thermodynamics of a family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A.

The C-terminal family 9 carbohydrate-binding module of xylanase 10A from Thermotoga maritima (CBM9-2) binds to amorphous cellulose, crystalline cellulose, and the insoluble fraction of oat spelt xylan. The association constants (K(a)) for adsorption to insoluble polysaccharides are 1 x 10(5) to 3 x 10(5) M(-1). Of the soluble polysaccharides tested, CBM9-2 binds to barley beta-glucan, xyloglucan, and xylan. CBM9-2 binds specifically to the reducing ends of cellulose and soluble polysaccharides, a property that is currently unique to this CBM. CBM9-2 also binds glucose, xylose, galactose, arabinose, cellooligosaccharides, xylooligosaccharides, maltose, and lactose, with affinities ranging from 10(3) M(-1) for monosaccharides to 10(6) M(-1) for disaccharides and oligosaccharides. Cellooligosaccharides longer than two glucose units do not bind with improved affinity, indicating that cellobiose is sufficient to occupy the entire binding site. In general, the binding reaction is dominated by favorable changes in enthalpy, which are partially compensated by unfavorable entropy changes.

Crystal structure of enteropathogenic Escherichia coli intimin-receptor complex.

Intimin and its translocated intimin receptor (Tir) are bacterial proteins that mediate adhesion between mammalian cells and attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli (EPEC) causes significant paediatric morbidity and mortality world-wide. A related A/E pathogen, enterohaemorrhagic E. coli (EHEC; O157:H7) is one of the most important food-borne pathogens in North America, Europe and Japan. A unique and essential feature of A/E bacterial pathogens is the formation of actin-rich pedestals beneath the intimately adherent bacteria and localized destruction of the intestinal brush border. The bacterial outer membrane adhesin, intimin, is necessary for the production of the A/E lesion and diarrhoea. The A/E bacteria translocate their own receptor for intimin, Tir, into the membrane of mammalian cells using the type III secretion system. The translocated Tir triggers additional host signalling events and actin nucleation, which are essential for lesion formation. Here we describe the the crystal structures of an EPEC intimin carboxy-terminal fragment alone and in complex with the EPEC Tir intimin-binding domain, giving insight into the molecular mechanisms of adhesion of A/E pathogens.

Structure and binding specificity of the second N-terminal cellulose-binding domain from Cellulomonas fimi endoglucanase C.

The 1,4-beta-glucanase CenC from Cellulomonas fimi contains two cellulose-binding domains, CBD(N1) and CBD(N2), arranged in tandem at its N-terminus. These homologous CBDs are distinct in their selectivity for binding amorphous and not crystalline cellulose. Multidimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy was used to determine the tertiary structure of CBD(N2) in the presence of saturating amounts of cellopentaose. A total of 1996 experimental restraints were used to calculate an ensemble of 21 final structures for the protein. CBD(Nu2) is composed of 11 beta-strands, folded into two antiparallel beta-sheets, with a topology of a jellyroll beta-sandwich. On the basis of patterns of chemical shift perturbations accompanying the addition of cellooligosaccharides, as well as the observation of intermolecular protein-sugar NOE interactions, the cellulose-binding site of CBD(N2) was identified as a cleft that lies across one face of the beta-sandwich. The thermodynamic basis for the binding of cellooligosaccharides was investigated using isothermal titration calorimetry and NMR spectroscopy. Binding is enthalpically driven and consistent with a structural model involving hydrogen bonding between the equatorial hydroxyls of the glucopyranosyl rings and polar amino acid side chains lining the CBD(N2) cleft. Affinity electrophoresis was used to determine that CBD(N2) also binds soluble beta-1,4-linked polymers of glucose, including hydroxyethylcellulose and beta-1,3-1,4-glucans. This study complements a previous analysis of CBD(N1) [Johnson, P. E., Joshi, M. D., Tomme, P., Kilburn, D. G., and McIntosh, L. P. (1996) Biochemistry 35, 14381-14394] and demonstrates that the homologous CBDs from CenC share very similar structures and sugar binding properties.

Characterization and affinity applications of cellulose-binding domains.

Cellulose-binding domains (CBDs) are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins. To date, almost 200 sequences can be classified in 13 different families with distinctly different properties. CBDs vary in size from 4 to 20 kDa and occur at different positions within the polypeptides; N-terminal, C-terminal and internal. They have a moderately high and specific affinity for insoluble or soluble cellulosics with dissociation constants in the low micromolar range. Some CBDs bind irreversibly to cellulose and can be used for applications involving immobilization, others bind reversibly and are more useful for separations and purifications. Dependent on the CBD used, desorption from the matrix can be promoted under various different conditions including denaturants (urea, high pH), water, or specific competitive ligands (e.g. cellobiose). Family I and IV CBDs bind reversibly to cellulose in contrast to family II and III CBDs which are in general, irreversibly bound. The binding of family II CBDs (CBD(Cex)) to crystalline cellulose is characterized by a large favourable increase in entropy indicating that dehydration of the sorbent and the protein are the major driving forces for binding. In contrast, binding of family IV CBDs (CBD(N1)) to amorphous or soluble cellulosics is driven by a favourable change in enthalpy which is partially offset by an unfavourable entropy change. Hydrogen bond formation and van der Waals interactions are the main driving forces for binding. CBDs with affinity for crystalline cellulose are useful tags for classical column affinity chromatography. The affinity of CBD(N1) for soluble cellulosics makes it suitable for use in large-scale aqueous two-phase affinity partitioning systems.

Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC.

The interaction of the N-terminal cellulose-binding domain, CBDN1, from Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investigated using NMR spectroscopy and calorimetry. CBDN1 binds a single calcium ion with an equilibrium association constant of approximately 10(5) M-1 at 35 degreesC and pH 6.0. Binding is exothermic (-42 +/- 2 kJ mol-1) under these conditions and is accompanied by a small negative change in heat capacity (DeltaCp = -0.41 +/- 0.16 kJ mol-1 K-1). From an NMR line shape analysis, the rate constants for calcium association and dissociation were found to be (5 +/- 2) x 10(7) s-1 M-1 and (4.5 +/- 0.6) x 10(2) s-1, respectively. The rapid association kinetics indicate that the calcium-binding site on CBDN1 is accessible and, to the first approximation, preformed. Based on patterns of chemical shift perturbations, and structural comparisons with the Bacillus sp. 1, 3-1,4-beta-glucanases, the backbone carbonyl oxygens of Thr8, Gly30, and Asp142 and a side chain carboxyl oxygen of Asp142 are postulated to form the calcium-binding site of CBDN1. Consistent with the calcium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosaccharide-binding cleft. The midpoint denaturation temperature of CBDN1 is increased by approximately 8 degreesC at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.

Stability and oligosaccharide binding of the N1 cellulose-binding domain of Cellulomonas fimi endoglucanase CenC.

Differential scanning calorimetry has been used to study the thermal stability and oligosaccharide-binding thermodynamics of the N-terminal cellulose-binding domain of Cellulomonas fimi beta-1,4-glucanase CenC (CBDN1). CBDN1 has a relatively low maximum stability (delta Gmax = 33 kJ/mol = 216 J/residue at 1 degree C and pH 6.1) compared to other small single-domain globular proteins. The unfolding is fully reversible between pH 5.5 and 9 and in accordance with the two-state equilibrium model between pH 5.5 and 11. When the single disulfide bond in CBDN1 is reduced, the protein remains unfolded at all conditions, as judged by NMR spectroscopy. This indicates that the intramolecular cross-link makes a major contribution to the stability of CBDN1. The measured heat capacity change of unfolding (delta Cp = 7.5 kJ mol-1 K-1) agrees well with that calculated from the predicted changes in the solvent accessible nonpolar and polar surface areas upon unfolding. Extrapolation of the specific enthalpy and entropy of unfolding to their respective convergence temperature indicates that per residue unfolding energies for CBDN1, an isolated domain, are in accordance with those found by Privalov (1) for many single-domain globular proteins. DSC thermograms of the unfolding of CBDN1 in the presence of various concentrations of cellopentaose were fit to a thermodynamic model describing the linkage between protein-ligand binding and protein unfolding. A global two-dimensional minimization routine is used to regress the binding enthalpy, binding constant, and unfolding thermodynamics for the CBDN1-cellopentaose system. Extrapolated binding constants are in quantitative agreement with those determined by isothermal titration calorimetry at 35 degrees C.

Thermostability and irreversible activity loss of exoglucanase/xylanase Cex from Cellulomonas fimi.

The activity of the beta-1,4-glycanase Cex (EC 3.2.1.91) from Cellulomonas fimi is investigated in connection with its industrial application in cellulose hydrolysis and its potential use in cellosaccharide synthesis. Catalytic activity measurements as a function of temperature, complemented with differential scanning calorimetry (DSC) data, are used to characterize the thermostability of the protein and the influence of interdomain interactions. The data suggest that the enzyme is irreversibly deactivated in one of two possible ways: (1) through a low-temperature route characterized by first-order kinetics; or (2) through a high-temperature route characterized by an initial reversible step followed by an irreversible step. Melting temperatures (Tm) of Cex and p-33 (the isolated catalytic domain of Cex) as estimated by DSC are 64.2 and 64.0 degrees C, respectively, suggesting that the binding and catalytic domains of the protein fold independently. Kinetic parameters (Km, kcat, and kcat/Km) of Cex for the hydrolysis of p-nitrophenyl beta-D-cellobioside (pNPC) were determined at temperatures ranging from 15 to 80 degrees C. As demanded by reversible mass-action thermodynamics, the Tm of Cex in the presence of excess ligand as determined from activity-temperature data is ca. 66.55 degrees C, more than 2 degrees C higher than the Tm for Cex under ligand-free conditions. The effect of temperature on the rate constant has been determined using Arrhenius plots. Combined with irreversible deactivation half-life data and DSC data, the results are used to evaluate a model, based on a theory developed by Hei et al. (1993), for predicting the time-dependent activity and active-state stability of the protein under a range of potential operating conditions.

Interaction of polysaccharides with the N-terminal cellulose-binding domain of Cellulomonas fimi CenC. 1. Binding specificity and calorimetric analysis.

The carbohydrate-binding specificity of the N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) was investigated using affinity electrophoresis, binding assays and microcalorimetry in parallel with NMR and difference ultraviolet absorbance spectroscopy [Johnson, P.E., Tomme, P., Joshi, M.D., & McIntosh, I., P. (1996) Biochemistry 35, 13895-13906]. Binding of CBDN1 on insoluble cellulose is distinctly different from other cellulose-binding domains. CBDN1 binds amorphous cellulose (phosphoric acid-swollen) with high affinity (Kr = 5.1 L g-1), binds Avicel weakly and does not bind highly crystalline bacterial or tunicin cellulose. Moreover, CBDN1 binds soluble cellooligosaccharides and beta-1,4-linked oligomers of glucose such as hydroxyethycellulose, soluble beta-1,3-1,4-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3-, or beta-1,6-polymers of glucose. This is the first report of a cellulose-binding domain with strong and specific affinity for soluble glycans. The thermodynamics for binding of CBDN1 to oligosaccharides, soluble glycans, and phosphoric acid-swollen cellulose were investigated by titration microcalorimetry. At least four beta-1,4-linked glucopyranosides are required to detect binding. For larger glucans, with five or more glucopyranoside units, the binding constants and standard free energy changes are virtually independent of the glucan chain length, indicating that cellopentaose completely fills the binding site. Binding is moderately strong with binding constants ranging from 3,200 +/- 500 M-1 for cellotetraose, to 25,000 +/- 3,000 M-1 for the larger sugars. The reactions are controlled by favorable standard free enthalpy changes which are compensated in a linear fashion by a significant decrease in entropy. A predominance of polar interactions such as hydrogen bonding together with van der Waals interactions provide the major driving forces for the binding event.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose-binding domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of 1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

Separation of amino-acid enantiomers using micellar-enhanced ultrafiltration.

Micellar-enhanced ultrafiltration (MEUF) is investigated as a large-scale technique for separating amino acid enantiomers. Specifically, L-5-cholesterol glutamate, a chiral ligand-exchange cosurfactant, is used together with a nonionic surfactant to form mixed micelles that preferentially bind D-phenylalanine over L-phenylalanine in the presence of copper(II). Operational selectivities as high as 4.2 are obtained. Potentiometric titrations using a water-soluble model compound similar to the chiral cosurfactant indicate that the ternary copper complex with phenylalanine has a stereoselectivity for the D enantiomer which is significantly smaller than that observed in the MEUF system. Thus, the selectivity of the chiral legend's local solvent and structural environment.

Structural and catalytic properties of enzymes in reverse micelles.

Structural and catalytic properties of two enzymes--alpha-chymotrypsin and horse liver alcohol dehydrogenase (LADH)--are studied in bis(2-ethylhexyl) sodium sulfosuccinate (AOT)-isooctane reverse-micelle solutions. Circular dichroism (CD) and electron paramagnetic resonance spectroscopy (EPR) studies show little change in alpha-chymotrypsin structure upon incorporation into reverse micelles. These structural properties explain, in part, the observed activity of these two enzymes in reverse micelles. alpha-Chymotrypsin retains activity in reverse micelles and, in some cases, displays enhanced activity. A sixfold increase in the turnover number was observed in w0 = 10 reverse micelles. LADH has low activity in reverse micelles compared to that in aqueous solution. At w0 = 70, the turnover number of LADH is 18% of the aqueous value. Active-site titrations show a decrease in active enzyme concentration for both enzymes upon incorporation into reverse micelles. Little change in the structure of both LADH and alpha-chymotrypsin is observed with change of water content in the reverse-micelle system.

The effect of aqueous surfactant solutions on alcohol dehydrogenase (LADH).

Alcohol dehydrogenase (LADH) was studied in aqueous solutions of surfactants to determine its structural and catalytic characteristics. Fluorescence, circular dichroism (CD), and electron paramagnetic resonance (ERP) techniques were used to study structural changes to the enzyme. The activity of LADH in catalyzing the oxidation of ethanol was investigated. Short-chain alkyl sulfonates and sulfates did not deactivate LADH or alter its structure. Longer and branched alkyl sulfates and sulfonates, as well as a cationic surfactant (CTAB), affected both LADH activity and conformation.

Mechanisms of protein solubilization in reverse micelles.

Solubilization properties of alpha-chymotrypsin and alcohol dehydrogenase (LADH) in reverse micelles are reported for three different solubilization techniques. The solubilization properties for these two proteins depend on the method used for protein addition. The addition of a dry protein powder to a reverse-micelle-containing organic phase does not appreciably solubilize the protein until the diameter of the reverse micelle is similar to that of the protein. However, when an aqueous protein solution is injected an organic phase, protein solubilization is not strongly dependent on micelle size. For chymotrypsin, multiple protein occupancy occurs at large micelle size, with as many as 11 chymotrypsin molecules solubilized in one reverse micelle. The solubilization of chymotrypsin using a phase-transfer technique with a positively charged surfactant follows the expected trend based on protein-surfactant electrostatic interactions. When a negatively charged surfactants is used for phase transfer, at low pH the solubilization data do not fit this electrostatic interaction mechanism. In this case, protein-surfactant aggregation may be occurring at the aqueous-organic interface.