Complete sequence and model for the A₂ subunit of the carotenoid pigment complex, crustacyanin

Abstract

The complete sequence has been determined for the A2 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus. The polypeptide chain is 174 residues long and is similar to proteins of the retinol-binding protein superfamily. Some regions of the sequence are most similar to the retinolbinding protein, P-lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin-binding protein. It is beginning to appear as though this superfamily of proteins, characterized by a similar gross structural framework, may be further subdivided into interrelated subclasses. Model building based on the coordinates of the known structure of human plasma retinolbinding protein and on empirical prediction algorithms has allowed the putative identification of side-chains which line the binding cavity. This pocket is larger than in retinol binding protein and P-lactoglobulin but does not allow the carotenoid to adopt a folded conformation. The amino acid composition of the pocket does not support a 'charge-shift'-type hypothesis to support the bathochromic shift phenomenon which takes place on interaction of the chromophore with the protein. Instead aromatic side-chains may play a prominent role.The blue colouration of the carapace of the lobster Homarus gammarus (L.) is provided by the astaxanthin (3,3'-dihydroxy-P,P-carotene-4,4'-dione) binding protein, acrustacyanin. Early interest in the carotenoprotein stemmed from similarities in its absorption characteristics to the visual pigment, rhodopsin [I]. Like rhodopsin, the absorption spectrum of the carotenoid (, , , 478 nm in acetone) is bathochromically shifted, without alteration in shape, by some 150 nm in the carotenoid-protein complex. Carotenoproteins occur ubiquitously in invertebrates where they may colour variously (red, purple, blue or green) eggs, ovary, blood or external surface (reviewed [2] and [3]).The physical and chemical properties of crustacyanin have been studied by a number of workers [4-91. The native pigment, a-crustacyanin, of molecular mass about 350 kDa is an aggregate of I6 apoprotein units of about 20 kDa, one astaxanthin molecule being bound/apoprotein monomer. The apoprotein consists of five electrophoretically distinct components which, from amino acid composition, size estimation and peptide mapping, fall into two main types: C1, Cz and Al (type I) and A2 and A3 (type 11), with C1 and Az predominating. Dimerization to form purple P-crustacyanin, also present in the lobster carapace, is brought about by association of two apoproteins, one of each type, with two carotenoid molecules; eight P-crustacyanin-sized units then associate to form a-crustacyanin. Correspondence to J . B. C . Findlay, Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, EnglandAbbreviations. RBP, retinol-binding protein; Pth-Xaa, phenylthiohydantoin derivative of amino acid Xaa; Hyd, Aro, Bas and Acd, hydrophobic, aromatic...

The C1subunit of α-crustacyanin: thede novophasing of the crystal structure of a 40 kDa homodimeric protein using the anomalous scattering from S atoms combined with direct methods

“…In common with the model of the C 1 /A 2 heterodimer (Keen et al, 1991a,b), the dimerization in the crystal is of a near face-toface nature. However, the residues (N-termini and loops GH, EF and DE of each monomer) and secondary-structural elements found at the dimer interface are markedly different from those predicted in the model dimer, which was based on the dimer found in the crystal structure of BBP (Huber et al, 1987;Keen et al, 1991b).…”

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

“…These shifts of 4.0 and 6.9 ppm are quite significant, but the crustacyanin dimer model of Keen et al [12,13] places no charged residues in the direct vicinity of the astaxanthin chromophore, including the 14,14' positions. If this is the case, other structural or electrostatic perturbations of astaxanthin in the ~-crustacyanin complex must be responsible for these downfield shifts.…”

“…Following the determination of the primary structures of the apoprotein subunits of crustacyanin, and the identification of significant homology between these sequences and those of other lipid-binding proteins, notably human plasma retinolbinding protein and fl-lactoglobulin, a model of the three-dimensional structure of the dimer, fl-crustacyanin, was constructed [12,13]. This model suggests that each subunit of the dimer contains 4 fl-pleated sheets that form a fl-barrel, and the astaxanthin molecule is positioned with one end in the apolar interior of the barrel.…”

The lipocalin protein family: structure and function

“…This includes several examples from arthropods : butterfly insecticyanin, grasshopper lazarillo [5], cockroach Bla g 4 protein [6] and lobster crustacyanin [7] ; and there is evidence to suggest that carotenoprotein lipocalins may also be present in species from the phylum Coelenterata [8]. Other calycins, FABPs and avidins are also present in both vertebrates and invertebrates, including Arthropoda and Coelentarata, but thus far there is only one example from prokaryotes, namely streptavidin.…”

“…Crustacyanin (meaning ' shell blue ') is the general name given to the carotenoprotein complex found in the epicuticle, or calcified outer layer, of the lobster carapace [7,84]. It acts as the dominant pigment of the lobster shell, giving rise to its characteristic blue colour.…”