Alain F. L. Creemers

(1)H and (13)C MAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin.

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven α-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete 1H and 13C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly 13C-labeled 11-cis-retinal. From the labeled pigment, 13C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment. The 1H chemical shifts were assigned by two-dimensional heteronuclear (1H-13C) dipolar correlation spectroscopy with phase-modulated Lee–Goldburg homonuclear 1H decoupling applied during the t1 period. The data indicate nonbonding interactions between the protons of the methyl groups of the retinylidene ionone ring and the protein. These nonbonding interactions are attributed to nearby aromatic acid residues Phe-208, Phe-212, and Trp-265 that are in close contact with, respectively, H-16/H-17 and H-18. Furthermore, binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH3-16 and CH3-17.

Synthesis of Isotopically Labelled L‐Phenylalanine and L‐Tyrosine

A synthetic route to stable-isotope-substituted L-phenylalanine is presented, which allows the introduction of 13C, 15N, and deuterium labels at any position or combination of positions. For labelling of the aromatic ring, a synthetic route to ethyl benzoate (or benzonitrile) has been developed, based on the electrocyclic ring-closure of a 1,6-disubstituted hexatriene system, with in situ aromatization by elimination of one (amino) substituent. Several important (highly isotopically enriched) synthons have been prepared, namely benzonitrile, benzaldehyde, ethyl benzoate, and ethyl diphenyloxyacetate. Labelled L-phenylalanines have been synthesized from both aromatic precursors by initial conversion into sodium phenylpyruvate and subsequent transformation of this intermediate into the L-α-amino acid by an enzymatic reductive amination reaction. In this manner, highly enriched phenylalanines are obtained on the 10-gram scale and with high enantiomeric purities (≥ 99% ee). The method has been validated by the synthesis of [1′-13C]-L-Phe and [2-D]-L-Phe. In addition, two methods are described for the introduction of isotopes into L-tyrosine starting from the isotopically enriched precursors benzonitrile and ethyl benzoate.

Synthesis of 13C-labeled carotenoids and retinoids

A three-part strategy has been developed to study molecular interactions in biological systems at the atomic level. First, isotopically labeled carotenoids and retinoids are prepared by organic total synthetic schemes with labels at predetermined atomic positions and combinations of positions. Subsequently, the labeled compounds are incorporated in the biological system. Finally, the system is studied by isotope sensitive spectroscopic techniques. In this paper, the synthesis of 10-fold 13 C-labeled retinal palmitate and b-carotene a- crustacyanin carotene for nutritional studies is discussed. Also, the scheme to label the end positions of astaxanthin and canthaxanthin with 13 C for spectroscopic investigations of a- crustacyanin with isotope labels in the chromophore is given. The synthesis of 10-methyl retinal is discussed, starting from isotopically labeled synthons obtained via schemes to 13 C- labeled natural retinal. Finally, the possibility for spectroscopic studies of caroteno and retino proteins via an expression of apoproteins by way of genetic techniques in the post-genomic era is discussed.

Determination of Torsion Angles in Membrane Proteins

Bacteriorhodopsin (bR) harnesses light energy to transport protons across the cell membrane of H. salinarium. Absorption of a photon by the protonated retinal chromophore initiates a cycle in which the chromophore releases a proton to an aspartate on the extracellular side and reprotonates from an aspartic acid on the cytoplasmic side. Vectorial proton transport depends on a switch in accessibility of the chromophore Schiff base nitrogen from the extracellular to cytoplasmic side. Changes in the retinal conformation are expected to be particularly important for understanding the pumping mechanism.