IDENTIFICATION OF A CONSERVED HYDROPHOBIC CLUSTER IN PARTIALLY FOLDEDBOVINE BETA-LACTOGLOBULIN AT PH 2

Background: NMR studies of denaturated states, both fully unfolded and partially folded, give insight into the conformations and interaction s formed during folding. Although the complete structural characteriza tion of partially folded proteins is a very difficult task, the identi fication of structured subsets, such as hydrophobic clusters, is of va lue in understanding the structural organization of such states. Here, we report the NMR characterization, in acidic conditions (pH 2), of a well-defined hydrophobic cluster localized in the core of bovine beta -lactoglobulin. Results: The existence of a small hydrophobic cluster present in the lipocalin protein family has been assessed on the basis of structural alignment and NMR data obtained for the partially folde d bovine beta-lactoglobulin. The presence of the cluster had been pred icted identifying those residues that are highly conserved in most mem bers of the family. An NMR study conducted at pH 2, where the protein exhibits a very stable beta-core together with disordered regions, rev eals the presence of NOEs among sidechains of 11 hydrophobic residues centered around Trp19 and pointing towards the interior of the protein . This buried cluster is found to be unusually stable at pH 2, not onl y at room temperature but also at 323K. Furthermore, conserved hydroph obic residues pointing towards the surface of the protein define a hyd rophobic surface patch located in a groove between the strands and the helix. Conclusions: 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 clu ster is present in these structures. We propose that the buried cluste r may represent the internal binding site as well and that the hydroph obic surface patch is involved in a second external binding site.