The binding of the carotenoid astaxanthin (AXT) in the protein multimacromolecular complex crustacyanin (CR) is responsible for the blue coloration of lobster shell. The structural basis of the bathochromic shift mechanism has long been elusive. A change in color occurs from the orange red of the unbound dilute AXT (max 472 nm in hexane), the well-known color of cooked lobster, to slate blue in the protein-bound live lobster state (max 632 nm in CR). Intriguingly, extracted CR becomes red on dehydration and on rehydration goes back to blue. Recently, the innovative use of softer x-rays and xenon derivatization yielded the threedimensional structure of the A 1 apoprotein subunit of CR, confirming it as a member of the lipocalin superfamily. That work provided the molecular replacement search model for a crystal form of the ␤-CR holo complex, that is an A1 with A3 subunit assembly including two bound AXT molecules. We have thereby determined the structure of the A3 molecule de novo. Lobster has clearly evolved an intricate structural mechanism for the coloration of its shell using AXT and a bathochromic shift. Blue͞purple AXT proteins are ubiquitous among invertebrate marine animals, particularly the Crustacea. The three-dimensional structure of ␤-CR has identified the protein contacts and structural alterations needed for the AXT color regulation mechanism.T he bathochromic shift of the absorption by astaxanthin (AXT) (3,3Ј-dihydroxy-␤,␤Ј-carotene-4,4Ј-dione; Fig. 1) in lobster crustacyanin (CR) has intrigued scientists for over 50 years (1-4). The visible absorption spectra of free dilute AXT, of ␤-and ␣-CR peak at 472 nm in hexane or 492 nm in pyridine, 580 and 632 nm, respectively (2). Detailed chemical studies involving reconstitution with natural and chemically synthesized carotenoids (4) have established the essential chemical characteristics required for this bathochromic shift effect, which arises from a reduced energy gap between ground and excited states. Keto groups are needed at positions 4 and 4Ј, conjugated with the polyene chain; methyl groups are needed at the central positions C20 and C20Ј; only E (all trans) isomers are bound to the protein; and no great variation in overall shape and size of the carotenoid is tolerated. The visible CD spectrum of ␤-CR, attributed to helical twisting of the chromophore, shows exciton splitting indicating that the two AXTs are proximal (2, 4). The lowered CϭC of AXT in the resonance Raman spectra of the CRs, indicative of increased electron delocalization in the electronic ground state, has been attributed to polarization of the -electron system by proximal charge groups or hydrogen bonds (3). These spectra argue against a large protein-induced distortion of the polyene chain, although minor changes to its geometry would be required to explain the spectra (3, 4). 13 C magic angle spinning NMR, together with Stark spectroscopy, gives support to an essentially symmetrical polarization of the AXT in CR with some asymmetry superimposed on the two halves of the chromo...

Biochemistry, biological crystallography, spectroscopy, solution X-ray scattering and microscopy have been applied to study the molecular basis of the colouration in lobster shell. This article presents a review of progress concentrating on recent results but set in the context of more than 50 years of work. The blue colouration of the carapace of the lobster Homarus gammarus is provided by a multimolecular carotenoprotein, alpha-crustacyanin. The complex is a 16-mer of five different subunits each binding the carotenoid, astaxanthin (AXT). A breakthrough in the structural studies came from the determination of the structure of beta-crustacyanin (protein subunits A1 with A3 with two shared bound astaxanthins). This was solved by molecular replacement using apocrustacyanin A1 as the search motif. A molecular movie has now been calculated by linear interpolation based on these two 'end-point' protein structures, i.e. apocrustacyanin A1 and A1 associated with the two astaxanthins in beta-crustacyanin, and is presented with this paper. This movie highlights the structural changes forced upon the carotenoid on complexation. In contrast, the protein-binding site remains relatively unchanged in the binding region, but there is a large conformational change occurring in a more remote surface-loop region. It is suggested here that this loop could be important in complexation of AXT and contributes to the spectral properties. Also presented here is the first observation of single-crystal diffraction of the full 'alpha-crustacyanin' complex comprising 16 protein subunits and 16 bound AXT molecules (i.e eight beta-crustacyanins) at 5 A resolution. Optimization of crystallization conditions is still necessary as these patterns show multiple crystallite character, however, 10 A resolution single-crystal diffraction has now been achieved. Provision of the new SRS MPW 10 and SRS MPW 14 beamline robotic systems will greatly assist in the surveying of the many alpha-crustacyanin crystallization trials that are being made. New solution X-ray scattering (SXS) measurements of beta- and alpha-crustacyanin are also presented. The beta-crustacyanin SXS data serve to show how the holo complex fits the SXS curve, whereas the apocrustacyanin A1 homodimer from the crystal data naturally does not. Reconstructions of alpha-crustacyanin were accomplished from its scattering-profile shape. The most plausible ultrastructure, based on a fourfold symmetry constraint, was found to be a stool with four legs. The latter is compared with published electron micrographs. A detailed crystal structure of alpha-crustacyanin is now sought in order to relate the full 150 nm bathochromic shift of AXT to that complete molecular structure, compared with the 100 nm achieved by the beta-crustacyanin protein dimer alone. Rare lobster colourations have been brought to attention as a result of this work and are discussed in an appendix.

The molecular basis of the camouflage colouration of marine crustacea is often provided by carotenoproteins. The blue colour of the lobster carapace, for example, is intricately associated with a multimacromolecular 16‐mer complex of protein subunits each with a bound astaxanthin molecule. The protein subunits of crustacyanin fall into two distinct subfamilies, CRTC and CRTA. Here, the crystal structure solution of the A1 protein of the CRTC subfamily is reported. The problematic nature of the structure solution of the CRTC proteins (both C1 and A1) warranted consideration and the development of new approaches. Three putative disulfides per protein subunit were likely to exist based on molecular‐­homology modelling against known lipocalin protein structures. With two such subunits per crystallographic asymmetric unit, this direct approach was still difficult as it involved detecting a weak signal from these sulfurs and suggested the use of softer X‐rays, combined with high data multiplicity, as reported previously [Chayen et al. (2000), Acta Cryst. D56, 1064–1066]. This paper now describes the structure solution of CRTC in the form of the A1 dimer based on use of softer X‐­rays (2 Å wavelength). The structure solution involved a xenon derivative with an optimized xenon LI edge signal and a native data set. The hand of the xenon SIROAS phases was determined by using the sulfur anomalous signal from a high‐multiplicity native data set also recorded at 2 Å wavelength. For refinement, a high‐resolution data set was measured at short wavelength. All four data sets were collected at 100 K. The refined structure to 1.4 Å resolution based on 60 276 reflections has an R factor of 17.7% and an Rfree of 22.9% (3137 reflections). The structure is that of a typical lipocalin, being closely related to insecticyanin, to bilin‐binding protein and to retinol‐binding protein. This A1 monomer or dimer can now be used as a search motif in the structural studies of the oligomeric forms α‐ and β‐crustacyanins, which contain bound astaxanthin molecules.