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...
A study on the concentration dependence of the modifications ensuing from thermal treatment of bovine /3-lactoglobulin was carried out by using a combination of techniques. Heat-induced changes in tertiary structure were monitored by intrinsic tryptophan fluorescence, while modifications in protein surface hydrophobicity were studied both during their occurrence and at equilibrium by using the fluorescent hydrophobic probe 1,8-anilinonaphthalenesulfonate. The association equilibria in the heated and cooled protein and the stabilization of aggregates by intermolecular disulfides were studied by gel permeation chromatography and nonreducing, denaturing electrophoresis.Results indicate that irreversible modification of the tertiary structure is not concentration dependent, while the temperature required for the occurrence of protein swelling, the initial step in the formation of associated forms of the protein, increases with the protein concentration. Stabilization of aggregates by intermolecular disulfides was dependent on concentration only at temperatures below 75 °C.
Gas-phase ions of protonated l-glutathione as native species, [GSH + H](+), and S-nitroso derivative, [GSNO + H](+), have been generated by electrospray ionization and probed via infrared multiple photon dissociation (IRMPD) action spectroscopy. Insight into the conformational landscape is gained from interpretation of the IR spectra aided by high-level theoretical calculations, which enables structural assignment disclosing both the site of protonation and the intramolecular hydrogen-bond network. Calculations yield the low-energy structures of [GSNO + H](+). A admixture of the four most stable ones (SN1, AN1, SN2, and AN2) is apt to account for the experimental IRMPD spectra obtained in both the 1000-2000 and the 3100-3700 cm(-1) spectral ranges. The most stable form of [GSNO + H](+), SN1, protonated at the amino group, presents a syn conformation at the S-N (partial) double bond and all peptidic carbonyls involved in (strong) C═O···H-N hydrogen bonds, so allowing closure of a C5 (β-strand), two C7 (γ-turn), and one C9-membered rings. An appreciable barrier to rotation of 43 kJ mol(-1) about the S-N bond is found to separate SN1 from the analogous anti isomer AN1, which lies only 0.70 kJ mol(-1) higher in free energy. Conformers obtained for [GSH + H](+) are very similar to the [GSNO + H](+) counterparts, indicating that the S-nitrosation motif does not affect significantly the geometry of the peptide. The observed ν(NO) signatures at 1622 and 1690 cm(-1), merged with other absorptions, are revealed by their sensitivity to (15)NO isotope labeling and by comparison with the IRMPD spectrum of native [GSH + H](+), providing a diagnostic probe for the S-nitrosation feature in natural peptides.
The binding motifs in the halide adducts with tyrosine ([Tyr + X], X = Cl, Br, I) have been investigated and compared with the analogues with 3-nitrotyrosine (nitroTyr), a biomarker of protein nitration, in a solvent-free environment by mass-selected infrared multiple photon dissociation (IRMPD) spectroscopy over two IR frequency ranges, namely 950-1950 and 2800-3700 cm. Extensive quantum chemical calculations at B3LYP, B3LYP-D3 and MP2 levels of theory have been performed using the 6-311++G(d,p) basis set to determine the geometry, relative energy and vibrational properties of likely isomers and interpret the measured spectra. A diagnostic carbonyl stretching band at ∼1720 cm from the intact carboxylic group characterizes the IRMPD spectra of both [Tyr + X] and [nitroTyr + X], revealing that the canonical isomers (maintaining intact amino and carboxylic functions) are the prevalent structures. The spectroscopic evidence reveals the presence of multiple non-covalent forms. The halide complexes of tyrosine conform to a mixture of plane and phenol isomers. The contribution of phenol-bound isomers is sensitive to anion size, increasing from chloride to iodide, consistent with the decreasing basicity of the halide, with relative amounts depending on the relative energies of the respective structures. The stability of the most favorable phenol isomer with respect to the reference plane geometry is in fact 1.3, -2.1, -6.8 kJ mol, for X = Cl, Br, I, respectively. The change in π-acidity by ring nitration also stabilizes anion-π interactions yielding ring isomers for [nitroTyr + X], where the anion is placed above the face of the aromatic ring.
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