Selective modification of Trp19 in beta-lactoglobulin by a new diazo fluorescence probe

To obtain the local information on the tryptophan domain in a protein, the design and synthesis of a new fluorescent probe, 1,7-bis(4-hydroxy-3-methoxyphenyl)-4-diazo-1,6-heptadiene-3,5-dione, is reported for the selective modification of tryptophan residues. The probe comprises a curcumin fluorophore and a diazo labeling group, whose spectroscopic properties are characterized. The diazo group may be catalytically degraded by transition metal complexes such as Rh2(OAc)4, generating an active rhodium carbenoid intermediate, which can react selectively with tryptophan residues. By the use of the carbene's intermolecular reactions, the tryptophan residue (Trp19) of beta-lactoglobulin may be modified with the diazo curcumin probe. Furthermore, slight secondary but larger tertiary structural changes are detected after Trp19 is modified, and the Trp19 modification produces a great effect on the binding of 8-anilino-1-naphthalenesulfonic acid and retinol. These results indicate that the Trp19 residue plays an essential role in the structure and stability of beta-lactoglobulin, and the specific modification of this residue may have a potential use in further elucidating the relationship between the structure and function of the protein.