NMR studies of retinoid-protein interactions: The conformation of [C-13]-beta-ionones bound to beta-lactoglobulin B

Purpose. Vitamin A (retinol) and its metabolites comprise the natural retin oids. While the biological action of these molecules are thought to be prim arily mediated by ca. 55 kDa nuclear retinoic acid receptors, a number of s tructurally similar 15-20 kDa proteins are involved in the transport, and p ossibly metabolism, of these compounds. The milk protein beta-lactoglobulin B (beta-LG) is an 18 kDa protein which binds retinol and may be involved i n oral delivery of retinol to neonates. beta-LG also binds drugs and other natural products and is of potential interest as a protective delivery vehi cle. Methods. To examine the conformation of the model retinoid beta-ionone both in solution and when bound to beta-LG, NMR and computational methods have been employed. Results. Taken together, NMR studies of beta-ionone in solution measuring s calar and dipolar coupling, as well as CHARMm calculations, suggest beta-io none prefers a slightly twisted 6-s-cis conformation. Isotope-edited NMR st udies of C-13-labeled beta-ionones bound to beta-LG, primarily employing th e HMQC-NOE experiment, suggest beta-ionone also binds to beta-LG in its 6-s -cis conformation. Conclusions. The methods employed here allow estimates of protein-bound lig and conformation. However, additional sites of ligand labeling wilt be nece ssary to aid in binding site localization.