The interactions of retinoids with retinol‐binding protein

Abstract

We have measured the pre-resonance Raman spectrum of retinal, retinoic acid and retinol in dilute CCl, solutions and when bound to the bovine-serum retinol-binding protein. The comparison reveals that the binding interaction does not involve any specific interactions of the head group and/ or the polyene chain with a particular protein residue. The data indicate hydrogen bonding of bound retinal's head-group oxygen to water, as well as some torsional angle change of its polyene chain upon binding.Retinol (vitamin A alcohol) circulates in blood bound to retinol-binding protein (RBP), a single polypeptide with a molecular mass of 21 kDa that has a single binding site for retinoids (Goodman, 1984). In the circulation, holo-RBP is complexed with another plasma protein, transthyretin. The three-dimensional structure of holo-RBP was determined by X-ray crystallography, and shows that it is composed of a single globular domain comprised of an N-terminal coil, a asheet core, an a-helix, and a C-terminal coil (Cowan et al., 1990;Newcomer et al., 1984). The core of RBP is composed of eight antiparallel p strands which are arranged in two stacked orthogonal p sheets. Retinol is encapsulated within the j? sheets with the p-ionone ring positioned deep within the /? barrel and the isoprene tail extending along the barrel axis to near the surface of the protein.The selectivity of RBP for binding various retinoids and other hydrophobic compounds has been hard to decipher from competition experiments despite intensive efforts. While some groups reported similar affinity of RBP for retinol, citral and p-ionone (Hase et al., 1976), and interpreted the results to indicate lack of a requirement for a fixed polyene chain length, others (Honvitz and Heller, 1974) reported no binding of p-ionone, deducing the opposite conclusion. The isomeric conformation of retinoids was reported to have no effect on binding in some cases (Horwitz and Heller, 1973) and to completely inhibit binding in others (Siegenthaler and Saurat, 1987). Similarly, conflicting reports were published regarding the necessity of a six-membered ring structure as well as a specific requirement of the polar group on the isoprene tail (Goodman and Raz, 1972;Hase et al., 1976; Honvitz and Heller, 1974 recent study of the thermodynamic parameters that contribute to the binding of retinol to RBP have shown that binding is stabilized purely by an increase in entropy and that the enthalpy of binding is approximately zero (Noy and Xu, 1990). These results were interpreted to indicate that binding is mainly stabilized by hydrophobic interactions, and that the hydroxyl moiety of retinol does not play a role in binding to RBP.An ideal method for study of chromophores embedded in proteins is resonance Raman spectroscopy. In this form of vibrational spectroscopy, the spectrum of a molecule is selectively probed by choosing an excitation light which overlaps the electronic absorption band of a chromophore. The resonantly enhanced Raman cross section is hence much larger than that of...

Interactions of all-trans-retinol and long-chain fatty acids with interphotoreceptor retinoid-binding protein

“…An increase in retinol fluorescence accompanies its association with other retinol-binding proteins, for example, RBP (Futterman & Heller, 1972;Cogan, 1976) and CRBP (Ong & Chytil, 1978; Noy & Blaner, 1991). The association of retinol with RBP is driven solely by hydrophobic interactions within the binding site (Noy & Xu, 1990a;Manor et al, 1993), while in CRBP, electrostatic interactions stabilize ligand-protein association (Jakoby et al, 1993). Thus, the fluorescence of retinol is found to increase following transfer from water to a binding site in varied binding-site environments 0 IRBP (1 µ ) was titrated with retinol in the presence of different fatty acids.…”