SPECTROSCOPIC CHARACTERIZATION OF ARRESTIN INTERACTIONS WITH COMPETITIVE LIGANDS - STUDY OF HEPARIN AND PHYTIC ACID-BINDING

A combination of intrinsic fluorescence and circular dichroic (CD) spe ctroscopy has been used to characterize the complexes formed between b ovine retinal arrestin and heparin or phytic acid, two ligands that ar e known to mimic the structural changes in arrestin attending receptor binding. No changes in the CD spectra were observed upon ligand bindi ng, nor did the degree of tryptophan fluorescence quenching change sig nificantly in the complexes. These data argue against any large-scale changes in protein secondary or tertiary structure accompanying ligand binding. The change in tyrosine fluorescence intensity was used to de termine the dissociation constants for the heparin and phytic acid com plexes of arrestin. The only change observed was a saturable diminutio n of tyrosine fluorescence signal from the protein. For both ligands, the data suggest two distinct binding interactions with the protein-a high-affinity interaction with K-d, between 200 and 300 nM, and a lowe r affinity interaction with K-d, between 2 and 8 mu M. Study of collis ional quenching of tyrosine fluorescence in free arrestin and the liga nd-replete complexes indicates that 10 of the 14 tyrosine residues of the protein are solvent-exposed in the free protein: this value drops to between 5 and 6 solvent-exposed residues in the high-affinity compl exes of the two ligands. These data suggest that ligand binding leads to direct occlusion of between 4 and 5 tyrosine residues on the solven t-exposed surface of the protein, but not to any large-scale changes i n protein structure. The large activation energy previously reported t o be associated with arrestin-receptor interactions may therefore refl ect localized movements of the N- and C-termini of arrestin, which are proposed to interact in the free protein through electrostatic intera ctions. Binding of the anionic ligands heparin, phytic acid, or phosph orylated rhodopsin may compete with the C-terminus of arrestin for the se electrostatic interactions, thus allowing the C-terminus to swing o ut of the binding region.