Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues.

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.

Concentration-dependent internalization of a cytokine/cytokine receptor complex in human hematopoietic cells.

Soluble steel factor (SF) is a potent stimulator of hematopoietic progenitor cell proliferation in vitro, and cytokine combinations that include SF can support extensive expansions of hematopoietic cells. Recently, we showed that very primitive progenitor cells from normal human bone marrow require exposure to very high concentrations of cytokines to maintain their primitive status while proliferating. These cells also display higher cell-specific cytokine uptake rates than more differentiated types of hematopoietic cells. As a first step toward identifying the mechanisms involved in mediating such cytokine dose-dependent effects, we have now investigated the kinetics of SF receptor (c-kit) internalization by human Mo7e cells exposed to different extracellular concentrations of soluble SF. Transfer of Mo7e cells to a higher concentration of SF caused an initially rapid downregulation of cell surface c-kit which was accompanied by a rapid depletion of extracellular SF. Confocal microscopy showed a concomitant increase in the number and intensity of intracellular c-kit aggregates. After the first 30 min, the cells continued to deplete SF from the medium but at a much slower rate. During this period, there was a gradual recovery of expression of c-kit on the cell surface. A mathematical analysis of bulk medium to cell-surface SF-mass transport indicated that the cytokine-depletion rates measured were not likely to have significantly depleted the SF concentration in the microenvironment of the cells. Taken together, these results underscore the importance of monitoring and appropriately regulating cytokine concentrations in hematopoietic cell expansion cultures. They may also help to explain the different biological responses exhibited by primitive hematopoietic cells exposed to different types and concentrations of cytokines for periods of days.

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.

Very high-level production and export in Escherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system.

A novel expression vector pTugA, previously constructed in our laboratory, was modified to provide kanamycin resistance (pTugK) and used to direct the synthesis of polypeptides as fusions with the C- or N-terminus of a cellulose binding domain which serves as the affinity tag in a novel secretion-affinity fusion system. Fed-batch fermentation strategies were applied to production in recombinant E. coli TOPP5 of the cellulose binding domain (CBD) from the Cellulomonas fimi cellulase Cex. The pTugK expression vector, which codes for the Cex leader sequence that directs the recombinant protein to the periplasm of E. coli, was shown to remain stable at very high-cell densities. Recombinant cell densities in excess of 90 g (dry cell weight)/L were achieved using media and feed solutions optimized using a 2(n) factorial design. Optimization of inducer (isophenyl-thio-beta-D-galactopyranoside) concentration and the time of induction led to soluble, fully active CBD(Cex) production levels in excess of 8 g/L.

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.