Structural features of transiently modified beta-lactoglobulin relevant to the stable binding of large hydrophobic molecules.
Binding sites for hydrophobic molecules on bovine beta-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 beta-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 beta-lactoglobulin present in solution at neutral pH and subdenaturing temperatures. Thus, the specificity of hydrophobes binding to beta-lactoglobulin may be modulated by simple physical treatment of the protein.
Lozinsky
Evgenia
E
Department of Chemistry, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva, 84105, Israel.
Iametti
Stefania
S
Barbiroli
Alberto
A
Likhtenshtein
Gertz I
GI
Kálai
Tamás
T
Hideg
Kálmán
K
Bonomi
Francesco
F
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D015335:Molecular Probes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism
D005227:Fatty Acids; D007782:Lactoglobulins; D015335:Molecular Probes; D014364:Tryptophan