Probing the retinol-binding site of bovine beta-lactoglobulin.
The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites. Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft. To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket. The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins. Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues. The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins. The blue shift indicates protonation of the Schiff base. The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol. These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.
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Department of Food Science, Cornell University, Ithaca, New York 14853.
Batt
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Sawyer
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D007700:Kinetics; D007782:Lactoglobulins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D014212:Tretinoin / Q000378:metabolism; D014801:Vitamin A / Q000378:metabolism*; D014961:X-Ray Diffraction
D017931:DNA Primers; D007782:Lactoglobulins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014801:Vitamin A; D014212:Tretinoin