Unravelling the structural chemistry of the colouration mechanism in lobster shell

Abstract

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.

Mechanisms of colour adaptation in the prawn Penaeus monodon

“…This interaction modifies the naturally red carotenoid, producing the diverse array of colours seen in the exoskeleton of crustaceans (reviewed in Chayen et al, 2003). During cooking, this interaction is disrupted, releasing the distinct red colouration of cooked seafood.…”

Combined biophysical techniques used to derive a model for alpha crustacyanin

“…to take into account EM density not considered by the EM software used. The calculated SAXS for the EM derived model is consistent, but not in perfect agreement, with the SAXS data [2]. Separate SAXS data modelling has been made both via ab initio and rigid body modelling methods.…”

“…With such an EM model there is always a risk of error such as from the staining and conformational heterogeneity producing artefacts in the single particle images. The development of this study will ideally involve cryoelectron microscopy or fresh crystals; even a crystal diffraction resolution of ~10 Å [2], and harnessing the EM model at 30 Å resolution reported here, would be a further next step forward in ultrastructure detail.…”

A novel function for a carotenoid: astaxanthin used as a polarizer for visual signalling in a mantis shrimp

“…When bound to specific proteins, thus forming astaxanthin-protein complexes, the absorbance spectra of the resulting carotenoproteins might be greatly shifted away from that of free astaxanthin (Zagalsky et al, 1990). The absorbance spectra of such carotenoproteins produce the colorful appearance of many crustaceans, including crabs and lobsters (Chayen et al, 2003). Similarly, incorporation of the ultraviolet-absorbing retinoid retinal into a diversity of opsin proteins can produce the variously absorbing visual pigments that underlie color vision (Okano et al, 1995;Yoshizawa, 1994).…”