TRYPTOPHAN-19 OF BETA-LACTOGLOBULIN, THE ONLY RESIDUE COMPLETELY CONSERVED IN THE LIPOCALIN SUPERFAMILY, IS NOT ESSENTIAL FOR BINDING RETINOL, BUT RELEVANT TO STABILIZING BOUND RETINOL AND MAINTAINING ITS STRUCTURE

Residue 19 of tryptophan in bovine beta-lactoglobulin (beta-LG) is the only invariant residue throughout the lipocalin superfamily having tw o characteristic features: binding ability for small hydrophobic molec ules and the unique beta-barrel three-dimensional structure. In this s tudy, we investigated whether this strictly conserved Trp-19 of beta-L G would be indispensable for its structure and function such as mainta ining the molecular structure and biological activity of beta-LG. Spec troscopic and enzymatic oxidation experiments on retinol bound to W19Y , in which Tyr was substituted for Trp-19, showed that Trp-19 was not critical for this binding, but was important for stably maintaining th e environment surrounding retinol and the bound retinol. An analysis, using four anti-beta-LG monoclonal antibodies as probes, revealed a st ructural change in region 20-29, but not in the reverse region of Trp- 19. A guanidine hydrochloride-induced unfolding study showed that the conformational stability of W19Y was greatly reduced by 6.9 kcal/mol c ompared to that of wild-type beta-LG. These facts indicated that Trp-1 9 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, there by conserving the bound retinol molecule.