Frataxin is an essential mitochondrial protein whose reduced expression causes Friedreich's ataxia (FRDA), a lethal neurodegenerative disease. It is believed that frataxin is an iron chaperone that participates in iron metabolism. We have tested this hypothesis using the bacterial frataxin ortholog, CyaY, and different biochemical and biophysical techniques. We observe that CyaY participates in iron-sulfur (Fe-S) cluster assembly as an iron-dependent inhibitor of cluster formation, through binding to the desulfurase IscS. The interaction with IscS involves the iron binding surface of CyaY, which is conserved throughout the frataxin family. We propose that frataxins are iron sensors that act as regulators of Fe-S cluster formation to fine-tune the quantity of Fe-S cluster formed to the concentration of the available acceptors. Our observations provide new perspectives for understanding FRDA and a mechanistic model that rationalizes the available knowledge on frataxin.

Heat-induced modifications in the tertiary and quaternary structure of P-lactoglobulin were followed at neutral pH for the protein at high temperature and for the protein that was heated and cooled. Fast changes in the environment of aromatic amino acids were apparent from near-ultraviolet-CD spectra of the heated protein and their intensity increased with increasing temperature. These modifications were irreversible only at temperatures higher than 65 -70°C. Addition of iodoacetamide during the heating/ cooling cycle greatly reduced the extent of irreversible modification of the tertiary structure of the protein.Reaction of the native P-lactoglobulin dimer with iodoacetamide or dithiobis(2-nitrobenzoic acid) was only observed upon heating at temperatures higher than 40 "C and resulted in progressive reaction of the unique sulfhydryl group in each of the two protein monomers. The sulfhydryl reagents induced release of a monomeric protein species that was no longer able to aggregate to the native dimeric form or to sequentially form polymers as found in the protein after heating at high temperature. Dimer dissociation was identified as the rate-limiting step in the reaction of P-lactoglobulin with sulfhydryl reagents. It occurred at temperatures much lower than those required for appreciable modification of the tertiary structure of the protein, and had an extremely high activation energy (E, = 213 kJ/mol). These results are compared with other published data, and a general mechanism for the formation of early reactive species in heat-treated P-lactoglobulin at neutral pH is proposed which stresses the relevant role of a highly hydrophobic, molten-globule-like free monomer that has an exposed sulfhydryl group on its surface.Keywords: p-lactoglobulin ; heat denaturation ; sulfhydryl groups.The globular protein P-lactoglobulin is found in the whey fraction of the milk of many mammals. In spite of numerous physical and biochemical studies, its function is not clearly understood [ l , 21. The crystal structure of bovine P-lactoglobulin has been determined and shows similarities to the plasma retinol-binding protein and the odorant-binding protein [3, 41. This finding suggests that the role of P-lactoglobulin may be connected with transport or accumulation of lipid-soluble biological components [5, 61.Refolding of the tertiary structure of &lactoglobulin from the chaotrope-denatured form has been investigated extensively at low pH, where the association of monomers into multimeric forms is minimal [7, 81 and refolding conforms to the moltenglobule hypothesis of intermediate formation in protein folding/ unfolding 191. The high stability of P-lactoglobulin at low pH has been explained by the strong stabilizing action of the two disulfide bonds present in its tertiary structure [2, lo]. The free, highly reactive -SH group of Cys121 in each monomer has been shown to be involved in intramolecular and intermolecular disulfide interchange with other -SH groups in treated milk [I 1 -131.Despite the large amount of structura...

Background: Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osborne’s three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat. Methods: Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osborne’s protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition. Results: The most important wheat allergens were the α-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients. Conclusions: The α-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.

Modifications in the exposure to the solvent of hydrophobic residues, changes in their organization into surface hydrophobic patches, and alterations in the dimerization equilibrium of beta-lactoglobulin upon thermal treatment at neutral pH 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 of beta-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.

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] The biosynthesis of iron-sulfur proteins is a multistep process involving a number of specialized proteins that mediate [FeS] cluster formation and delivery to acceptor proteins (reviewed in Refs. 1-3). One of the key features of the current view of the pathway is the proposed participation of protein scaffolds that function as the initial sites for formation of transient [FeS] clusters; these molecular scaffolds may serve to guide cluster assembly, protect nascent clusters in the cellular environment, and/or assist in cluster transfer to acceptor proteins. The concept of scaffold-mediated metallocluster formation emerged from studies on the nitrogenase MoFe protein (4 -6), and initial formation of [FeS] clusters on scaffold proteins was first proposed for NifU, a protein required for the biogenesis of [FeS] clusters in the nitrogenase Fe-protein (7). Subsequent studies of [FeS] cluster formation on other proteins implicated in ironsulfur protein biogenesis, the NifU-related protein IscU/Isu (8, 9), IscA/Isa (10, 11), and SufA (12), led to the suggestion that these proteins may also function as specific scaffolds for preassembly of [FeS] clusters.In vitro studies demonstrating transfer of preformed scaffold- [FeS] complexes from NifU (13) (21, 22) show that cysteine residues presumed to be involved in binding to iron atoms of the clusters are located at the surface of the apo-forms of the scaffold proteins. The scaffold-bound [FeS] clusters are therefore likely to be at least partially exposed to solvent and accessible for transfer to acceptor proteins. Little is known about how the clusters are released, but there is some evidence for a direct transfer involving scaffold-acceptor complexes. Studies employing a modified form of human Isu containing a 57 Fe-labeled cluster showed that the label did not exchange with ferrous sulfate present in solution during cluster transfer to apo-ferredoxin (15), and studies with Escherichia coli IscA and SufA showed that the iron chelator bathophenanthroline sulfonate does not interfere with cluster transfer to apo-BioB (12). These findings are consistent with a mechanism in which transfer occurs without a significant degree of cluster disassembly and reassembly and suggest that clusters may be captured immediately upon release from the scaffold protein or may be transferred directly in a scaffoldacceptor protein complex. Evidence that complex formation can occur has been obtained for IscA-ferredoxin and SufA-BioB pairs using affinity chromatography (16,19) and for Isu-and Isa-ferredoxin pairs using chemical cross-linking (9, 11). However, the nature of the scaffold-acceptor interactions has not been characterized, and it has not been firmly established that complex formation is required for cluster t...