Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with β-cyclodextrin

We have used UV difference spectroscopy and fluorescence spectroscopy to study the perturbation by β-cyclodextrin of tyrosyl or tryptophyl residues located at each of the 10 variable consensus contact positions in the third domain of turkey ovomucoid. The goal was to monitor the accessibility of the side chain rings of these residues when located at these positions. The results indicated that the tyrosyl or tryptophyl rings are most highly exposed when located in the P1 position followed by the P4 position. It was possible to determine the association constants for β-cyclodextrin binding at these positions. When located at the P2, P5, P6 and P3′ positions, the rings of the tyrosyl or tryptophyl residues were exposed but less so than at the P1 or P4 positions. By contrast, when located at the P1′, P2′, P14′ and P18′ positions, the tyrosyl or tryptophyl residues were insufficiently exposed to be perturbed by β-cyclodextrin, although they reacted positively to dimethyl sulfoxide solvent perturbation. These findings indicate that β-cyclodextrin perturbation provides a convenient way to detect highly exposed tyrosyls or tryptophyls in proteins. Furthermore, we evaluated the ability of β-cyclodextrin to inhibit the interaction of turkey ovomucoid third domain variants with different P1 residues. The results showed that the presence of β-cyclodextrin had little effect on the association constant when the P1 residue was a glycyl residue, but greatly decreased the association constant when the P1 residue was a tyrosyl or tryptophyl residue. Thus, β-cyclodextrin may be used to selectively modulate the interaction between proteinase inhibitors and their cognate enzymes.