Frans Jos Jansen

Protein‐chromophore interactions in α‐crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus a study by 13C magic angle spinning NMR

MAS (magic angle spinning) 13C NMR has been used to study protein‐chromophore interactions in α‐crustacyanin, the blue astaxanthin‐binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14′ or 15,15′ positions. Two signals are seen for α‐crustacyanin containing [14,14′‐13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With α‐crustacyanin containing [15,15′‐13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in α‐crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176–191]. The splitting of the 14 and 14′ signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.

The spectroscopic and photochemical properties of locked-15,15′-cis-spheroidene in solution and incorporated into the reaction center of Rhodobacter sphaeroides R-26.1

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15′-cis-spheroidene, were studied by absorption, fluorescence, circular dichroism, fast transient absorption and electron spin resonance spectroscopies in solution and after incorporation into the reaction center of Rhodobacter (Rb.) sphaeroides R-26.1. HPLC purification of the synthetic molecule reveals the presence of several di-cis geometric isomers in addition to the mono-cis isomer of locked-15,15′-cis-spheroidene. In solution, the absorption spectrum of the purified mono-cis sample was red-shifted and showed a large cis-peak at 351 nm compared to unlocked all-trans spheroidene. Molecular modeling and semi-empirical calculations reveal how geometric isomerization and structural factors affect the room temperature spectra. The spectroscopic studies of the purified locked-15,15′-mono-cis molecule in solution reveal a more stable manifold of excited states compared to the unlocked spheroidene. Reaction centers of Rb. sphaeroides R-26.1 in which the locked-15,15′-cis-spheroidene was incorporated show no difference in either the spectroscopic properties or photochemistry compared to reaction centers in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 reaction centers which naturally contain spheroidene. The data suggest that the natural selection of a cis-isomer of spheroidene for incorporation into native reaction centers of Rb. sphaeroides wild type strain 2.4.1 is more determined by the structure or assembly of the reaction center protein than by any special quality of the cis-isomer of the carotenoid that would affect its ability to participate in triplet energy transfer or carry out photoprotection.

The mechanism of the colour shift of astaxanthin in α-crustacyanin as investigated by 13 C MAS NMR and specific isotope enrichment

By selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in a-crustacyanin, the major blue astaxanthin binding carotenoprotein complex from the carapace of the lobster Homarus gammarus, have been analysed. 13C Magic Angle Spinning (MAS) NMR spectra were obtained after reconstitution with astaxanthins labelled in the centre of the molecule or at the two keto groups. The MAS data reveal electrostatic polarizations of the conjugated chain. In addition, solid state NMR results for pure unlabelled astaxanthin can be compared with natural abundance I3C MAS data for canthaxanthin and p-carotene, to address the effect of the ring functionalities on the electronic properties of the polyene chain. Quantum chemical calculations were performed to reconcile the MAS data with one of several simple and straightforward mechanisms for the colour shift. The results point towards a colour shift mechanism in which the astaxanthin may be doubly charged, possibly by a double protonation of the two ring keto groups.

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.

Synthesis and Characterization of all‐E‐(4,4′‐13C2)‐Astaxanthin Strategies for Labelling the C15‐End Groups of Carotenoids

The all-E isomer of (4,4′-13C2)astaxanthin (1a) has been prepared by total synthesis starting from commercially available 99% 13C enriched acetonitrile. The labelled astaxanthin was obtained in high purity and with high isotope incorporation. For this synthesis, the C15 + C10 + C15 strategy was used. The central C10-synthon, 2,7-dimethylocta-2,4,6-triene-1,8-dial (3), was coupled with 13C-enriched C15-phosphonium salt 2a. The new synthetic scheme for the preparation of the C15-phosphonium salt is discussed in this paper; the same scheme can be used to label all positions and combinations of positions of the C15-phosphonium salt.

Quantum chemical modelling and spectroscopic studies of the protein-astaxanthin interactions in α-crustacyanin

Carotenoproteins, commonly found in marine invertebrate animals, are responsible for green, purple or blue colours. In contrast, free carotenoid exhibit yellow, orange or red colours. An example is provided by the lobster Homarus gammarus. Astaxanthin, which is responsible for the colour, is deep-blue when the lobster is alive and becomes bright orange-red after it is being cooked. The nature of the interactions by which the protein causes the large bathochromic shift of ca 150 nm in the absorption spectrum of astaxanthin is still a matter of debate. This energy shift is comparable to the opsin shift in the visual retinal pigments and bacteriorhodopsin. In these complexes retinal is covalently bound to the protein as a protonated Schiff base whereas in carotenoproteins, the interactions are non-covalent. Previous studies indicated that the β-ring keto groups are essential for the formation of complexes, and favoured a polarisation mechanism [1].