Structural Features of Transiently Modified Beta-Lactoglobulin Relevant to the Stable Binding of Large Hydrophobic Molecules |
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Authors: | Evgenia Lozinsky Stefania Iametti Alberto Barbiroli Gertz I Likhtenshtein Tamás Kálai Kálmán Hideg Francesco Bonomi |
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Institution: | (1) Department of Chemistry, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva, 84105, Israel;(2) Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Via G. Celoria, 2, 20133 Milan, Italy;(3) Department of Organic and Medicinal Chemistry, University of Pécs, PO Box 99, H-7602 Pécs, Hungary |
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Abstract: | Binding sites for hydrophobic molecules on bovine β-lactoglobulin, and their susceptibility to temperature, were studied by
using various spectroscopic probes. Binding of probes carrying a single fluorophore moiety, a single nitroxide moiety, or
both moieties on the same molecule, was followed by EPR and fluorescence. The presence of a fatty acid side chain in the dual
probes was found to be required for binding to
β-lactoglobulin. Binding occurred only after the protein was heated at temperatures below the threshold for its irreversible
denaturation. Binding became extremely tight and stable upon cooling of the protein–probe mixture. Comparison among the various
probes suggests that multiple binding sites for hydrophobes are present in the native protein, and in the partially—and reversibly—modified
form of β-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes
binding to β-lactoglobulin may be modulated by simple physical treatment of the protein. |
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Keywords: | β -lactoglobulin dual fluorescence-spin probes hydrophobic binding sites temperature-induced protein modification |
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