Identification of a conserved hydrophobic cluster in partially folded bovine β-lactoglobulin at pH 2

Abstract

The detected buried cluster most likely plays an important role in bovine beta-lactoglobulin stability. The analysis of five structurally related proteins reveals that the same extended cluster is present in these structures. We propose that the buried cluster may represent the internal binding site as well and that the hydrophobic surface patch is involved in a second external binding site.

Equilibrium unfolding CD studies of bovine ?-lactoglobulin and its 14-52 fragment at acidic pH

“…It is in fact known that very stable ␤ structures can often persist at high concentrations of GdnHCl or urea and may behave as folding initiation sites. Indeed our nmr data showed the existence of a highly structured ␤-core at acidic pH 3,4 and the persistence of the ␤ structure at 37°C and 5M urea (Molinari et al, manuscript in preparation).…”

“…Hence the formation of ␤ structures is not a common property in short peptides in SDS below the critical micellar concentration. For instance, the modified peptide Y(EAAAK) 3 A, showing ␣-helical propensity, is indeed ␣-helical in all solvents, even in 2 mM SDS and in aqueous solution. 29 Besides, the CD spectrum of TRM, a peptide with ␤␣␤ propensity, is not typical of a ␤-strand in nonmicellar SDS; it is rather remi- niscent of an ␣/␤ protein, with little helix and a mixture of ␤-strands, turns, and other conformations, as expected for random structures.…”

“…Its topology consists of 9 antiparallel ␤-strands and 1 ␣-helix as determined by x-ray 1 and nmr studies [2][3][4][5] obtained from the protein in physiological conditions and at acidic pH, respectively. In spite of its being a ␤-barrel protein, several secondary structure predictions have shown that bovine ␤-LG has a high propensity for ␣-helical conformation.…”

β-Lactoglobulin Binds Palmitate within Its Central Cavity

“…4 -6) because of its size, convenience (ϳ3 g/liter of milk), and stability. The ruminant protein is normally a dimer at room temperature and physiological pH, although at pH values below 3 and above 8, it dissociates into monomers, for which the structure at low pH is similar to that at pH 7 (7). A further reason for studying ␤-Lg is its involvement in the loss of functionality during the heat processing of milk (8).…”

Molecular mobility and oxygen permeability in amorphous β-lactoglobulin films

“…with longer aliphatic chains (palmitate and laurate) are found to display higher affinities for the protein (Ragona et al, 2000;Collini et al, 2003). Two potential binding sites have been postulated for small hydrophobic molecules, an interior cavity within the beta barrel and a surface cleft located in a groove between the helix and the barrel Dufour et al, 1994;Ragona et al, 1997). However, bovine β-Lg displays only one high affinity-binding site for palmitate with an apparent dissociation constant of 1x10 A very low-binding specificity of caprylic acid to β-Lg was observed, possibly due to its minor sterical hindrance, thus conferring a large conformational freedom within a cavity that preferentially hosts the endogenous longer-chain fatty acids .…”