Francesco Bonomi

Reversible and irreversible modifications ofβ-lactoglobulin upon exposure to heat

Modifications in the exposure to the solvent of hydrophobic residues, changes in their organization into surface hydrophobic patches, and alterations in the dimerization equilibrium ofβ-lactoglobulin upon thermal treatment at neutralpH were studied. Exposure of tryptophan residues was temperature dependent and was essentially completed on the time scale of seconds. Reorganization of generic hydrophobic protein patches on the protein surface was monitored through binding of 1,8-anilinonaphthalenesulfonate, and was much slower than changes in tryptophan exposure. Different phases in surface hydrophobicity changes were related to the swelling and the subsequent collapse of the protein, which formed a metastable swollen intermediate. Heat treatment ofβ-lactoglobulin also resulted in the formation of soluble oligomeric aggregates. The aggregation process was studied as a function of temperature, demonstrating that (i) dimer dissociation was a necessary step in a sequential polymerization mechanism and (ii) cohesion of hydrophobic patches was the major driving force for aggregation.

Reduction of immunoreactivity of bovine β-lactoglobulin upon combined physical and proteolytic treatment

Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin before, during and after treatment at 600 MPa and pH 6.8 for 10 min at 30, 37 and 44 degrees C. The extent of beta-lactoglobulin hydrolysis under pressure was noticeably higher than at atmospheric pressure, particularly when chymotrypsin was used. Addition of proteases at ambient pressure to previously pressure-treated beta-lactoglobulin gave only a modest increase in proteolysis with respect to the untreated protein. Products of enzyme hydrolysis under pressure were separated by reverse-phase HPLC, and were found to be different from those obtained at atmospheric pressure when chymotrypsin was used. The residual immunochemical reactivity of the products of combined pressure-enzyme treatment was assessed on the unresolved hydrolysates by ELISA tests using polyclonal and monoclonal antibodies, and on individual hydrolytic fractions by Western Blotting using sera of paediatric patients allergic to whey proteins in cow milk. The immunoreactivity of the whole hydrolysates was related to their content of residual intact beta-lactoglobulin, and no immunochemical reactivity was found for all the products of chymotrypsin hydrolysis under pressure. The results indicate that chymotrypsin effectively hydrolysed hydrophobic regions of beta-lactoglobulin that were transiently exposed during the pressure treatments and that were not accessible in the native protein or in the protein that had been previously pressure treated.

Kinetic and immobilization studies on fungal glycosidases for aroma enhancement in wine

Abstract The biochemical properties of a commercial glycosidase from Aspergillus niger (Cytolase PCL5, Genencor) were investigated. The product contains β-glucosidase, α-arabinosidase, and α-rhamnosidase activities in a ratio considered suitable for aroma enhancement in wine-making. A kinetic study of these three activities was carried out, which included determination of kinetic constants; dependence of enzyme activity and stability on pH and temperature; and enzyme inhibition by glucose, fructose, glycerol, and sulfurous anhydride. These glycosidase activities were immobilized to a solid carrier with the aim of developing a continuous process for wine aroma enhancement. Immobilization was best achieved with silanized bentonite as the solid, activated carrier, with bound glutaraldehyde as the reactive arm at a protein:carrier ratio of 0.11. Immobilization reaction was carried out for 48 h at pH 4 and 30°C. Under these conditions, 16 units of β-glucosidase, 2 units of α-arabinosidase, and 4.5 units of α-rhamnosidase per gram of bentonite were immobilized.

Studies on the Mechanism of Catalysis of Iron-Sulfur Cluster Transfer from IscU[2Fe2S] by HscA/HscB Chaperones

The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU(2)[2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperone-catalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU(2)[2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU(2)[2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU(2)[2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU(2)[2Fe2S] complex during the T --> R transition of HscA accompanying ATP hydrolysis.

Multiple Turnover Transfer of [2Fe2S] Clusters by the Iron-Sulfur Cluster Assembly Scaffold Proteins IscU and IscA

IscU/Isu and IscA/Isa (and related NifU and SufA proteins) have been proposed to serve as molecular scaffolds for preassembly of [FeS] clusters to be used in the biogenesis of iron-sulfur proteins. In vitro studies demonstrating transfer of preformed scaffold-[FeS] complexes to apoprotein acceptors have provided experimental support for this hypothesis, but investigations to date have yielded only single-cluster transfer events. We describe an in vitro assay system that allows for real-time monitoring of [FeS] cluster formation using circular dichroism spectroscopy and use this to investigate de novo [FeS] cluster formation and transfer from Escherichia coli IscU and IscA to apo-ferredoxin. Both IscU and IscA were found to be capable of multiple cycles of [2Fe2S] cluster formation and transfer suggesting that these scaffold proteins are capable of acting “catalytically.” Kinetic studies further showed that cluster transfer exhibits Michaelis-Menten behavior indicative of complex formation of holo-IscU and holo-IscA with apoferredoxin and consistent with a direct [FeS] cluster transfer mechanism. Analysis of the dependence of the rate of cluster transfer, however, revealed enhanced efficiency at low ratios of scaffold to acceptor protein suggesting participation of a transient, labile scaffold-[FeS] species in the transfer process.

Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His69 residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (∼3 versus ∼5 m, respectively), and the mutant protein had a melting temperature ∼10–15 °C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 m urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.

Facilitated Transfer of IscU–[2Fe2S] Clusters by Chaperone-Mediated Ligand Exchange

The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.

Structure–quality relationship in commercial pasta: A molecular glimpse

Presence and stability of a protein network was evaluated by fluorescence spectroscopy, by protein solubility studies, and by assessing the accessibility of protein thiols in samples of commercial Italian semolina pasta made in industrial plants using different processes. The pasting properties of starch in each sample were evaluated by means of a viscoamylograph. Magnetic resonance imaging (MRI) was used to evaluate water distribution and water mobility in dry pasta, and at various cooking times. The molecular information derived from these studies was related to sensory indices, indicating that protein reticulation was dependent on the process conditions, which affected water penetration, distribution, and mobility during cooking. Products with a crosswise gradient of water mobility once cooked had the best sensory scores at optimal cooking time, whereas products with a less compact protein network performed better when slightly overcooked.

Structural changes of soy proteins at the oil-water interface studied by fluorescence spectroscopy.

Fluorescence spectroscopy was used to acquire information on the structural changes of proteins at the oil/water interface in emulsions prepared by using soy protein isolate, glycinin, and β-conglycinin rich fractions. Spectral changes occurring from differences in the exposure of tryptophan residues to the solvent were evaluated with respect to spectra of native, urea-denatured, and heat treated proteins. The fluorescence emission maxima of the emulsions showed a red shift with respect to those of native proteins, indicating that the tryptophan residues moved toward a more hydrophilic environment after adsorption at the interface. The heat-induced irreversible transitions were investigated using microcalorimetry. Fluorescence spectroscopy studies indicated that while the protein in solution underwent irreversible structural changes with heating at 75 and 95°C for 15 min, the interface-adsorbed proteins showed very little temperature-induced rearrangements. The smallest structural changes were observed in soy protein isolate, probably because of the higher extent of protein-protein interactions in this material, as compared to the β-conglycinin and to the glycinin fractions. This work brings new evidence of structural changes of soy proteins upon adsorption at the oil water interface, and provides some insights on the possible protein exchange events that may occur between adsorbed and unadsorbed proteins in the presence of oil droplets.

Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin

Abstract Rubrerythrin (Rr) is the trivial name given to a non-heme iron protein of unknown function which has been found in anaerobic sulfate-reducing bacteria. Rr is unique in containing both rubredoxin-type FeS4 and diiron-oxo sites in the same protein. The results described here demonstrate for the recombinant protein that: (a) Rr catalyzes oxidation of Fe2+ to Fe3+ by O2, i.e., Rr has ferroxidase activity, (b) both FeS4 and diiron domains of the Rr protein are required for ferroxidase activity, (c) with excess Fe2+ and O2 the initial rate of this oxidation appears to be first order in [Rr] and independent of starting [Fe2+] above 30 μM, (d) the Fe3+ is produced in a form which is capable of rapid incorporation into the iron-binding site of ovotransferrin, and (e) the ferroxidase activity of Rr is comparable to that of published ferroxidase activities of apoferritins on a subunit basis. Ferroxidase activity of Rr was monitored either by the rate of increase in absorbance at 315 nm (which lies near an isosbestic point for oxidized and reduced Rr) or by using apoovotransferrin as Fe3+ acceptor, and measuring the rate and extent of diferric transferrin formation at 460 nm. No polyironoxyhydroxide aggregates appeared to associate with Rr after the ferroxidase reaction. A truncated form of Rr containing only the diiron domain had little or no ferroxidase activity. Rr could function as one component of a set of enzymes which channels the reaction products of O2 and Fe2+ onto a non-toxic pathway during transient exposure of the bacteria to air.