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 two characteristic features: binding ability for small hydrophobic molecules and the unique beta-barrel three-dimensional structure. In this study, we investigated whether this strictly conserved Trp-19 of beta-LG would be indispensable for its structure and function such as maintaining the molecular structure and biological activity of beta-LG. Spectroscopic 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 the environment surrounding retinol and the bound retinol. An using four anti-beta-LG monoclonal antibodies as probes, revealed a structural 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 compared to that of wild-type beta-LG. These facts indicated that Trp-19 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, thereby conserving the bound retinol molecule.
Katakura
Y
Y
Department of Agricultural Chemistry, University of Tokyo, Japan.
Totsuka
M
M
Ametani
A
A
Kaminogawa
S
S
D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry; D002942:Circular Dichroism; D004795:Enzyme Stability; D007782:Lactoglobulins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054835:Lipocalin 1; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012471:Salivary Proteins and Peptides / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*; D014801:Vitamin A / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D007782:Lactoglobulins; D054835:Lipocalin 1; D012471:Salivary Proteins and Peptides; D014801:Vitamin A; D014364:Tryptophan