Peter F. Zagalsky

The molecular basis of the coloration mechanism in lobster shell: β-Crustacyanin at 3.2-Å resolution

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 (λmax 472 nm in hexane), the well-known color of cooked lobster, to slate blue in the protein-bound live lobster state (λmax 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 three-dimensional structure of the A1 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 β-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. Blue/purple AXT proteins are ubiquitous among invertebrate marine animals, particularly the Crustacea. The three-dimensional structure of β-CR has identified the protein contacts and structural alterations needed for the AXT color regulation mechanism.

CCD video observation of microgravity crystallization: apocrustacyanin C1

Abstract Apocrustacyanin C 1 has been crystallized in the vapour-diffusion apparatus of ESAs Advanced Protein Crystallization Facility (APCF) on-board the NASA space shuttle STS-65 International Microgravity Laboratory-2 (IML-2) mission. Crystal growth was monitored by black and white CCD observation at time intervals throughout the experiment. The resulting crystals displayed a motion within the hanging drop that is attributed to Marangoni convection effects. The images also show a “halo” effect around the growing crystals which can be attributed to the presence of depletion zones i.e. solution regions which are depleted of this coloured protein.

Protein Crystal Movements and Fluid Flows During Microgravity Growth

The growth of protein crystals suitable for X–ray crystal structure analysis is an important topic. The methods of protein crystal growth are under increasing study whereby different methods are being compared via diagnostic monitoring including charge coupled device (CCD) video and interferometry. The quality (perfection) of protein crystals is now being evaluated by mosaicity analysis (rocking curves) and X–ray topographic images as well as the diffraction resolution limit and overall data quality. Choice of a liquid—liquid linear crystal–growth geometry and microgravity can yield a spatial stability of growing crystals and fluid, as seen in protein crystallization experiments on the uncrewed platform EURECA. A similar geometry used within the Advanced Protein Crystallization Facility (APCF) onboard the crewed shuttle missions SpaceHab–01 and IML–2, however, has shown by CCD video some lysozyme crystal movement through the mother liquor. Moreover, spurts and lulls of growth of a stationary lysozyme protein crystal that was probably fixed to the crystal–growth reactor wall suggests g–jitter stimulated movement of fluid on IML–2, thus transporting new protein to the growing crystal faces. In yet another study, use of a hanging drop vapour diffusion geometry on the IML–2 shuttle mission showed, again via CCD video monitoring, growing apocrustacyanin C1 protein crystals executing near cyclic movement, reminiscent of Marangoni convection flow of fluid, the crystals serving as ‘markers’ of the fluid flow. These observations demonstrated that the use of vapour diffusion geometry did not yield spatially stable crystal position or fluid conditions for a solely protein diffusive regime to be realized. Indeed mosaicity evaluation of those vapour diffusion–grown apocrustacyanin C1 crystals showed inconsistent protein crystal quality, although the best crystal studied was microgravity grown. In general, realizing perfect conditions for protein crystal growth, of absence of movement of crystal or fluid, requires not only the correct choice of geometry but also the avoidance of low–frequency (≲5Hz) g–jitters. A review is given here of existing results and experience over several microgravity missions. Some comment is given on gel protein crystal growth in attempts to ‘mimic’ the benefits of microgravity on Earth. Finally, the recent new results from our experiments on the shuttle mission LMS are described. These results include CCD video as well as interferometry during the mission, followed, on return to Earth, by reciprocal space mapping at the NSLS, Brookhaven and full X–ray data collection on LMS and Earth control lysozyme crystals. Diffraction data recorded from LMS and ground control apocrustacyanin C1 crystals are also described.

Quaternary structure of alpha-crustacyanin from lobster as seen by small-angle X-ray scattering.

The structure of α‐crustacyanin, the blue carotenoprotein of lobster (Homarus gammarus) carapace, has been investigated for the first time using small‐angle X‐ray scattering. In this paper, we have determined the dimensions of this protein composed of eight heterodimeric subunits of β‐crustacyanin. Analysis of the scattering spectra and estimation of the shape of α‐crustacyanin show that the protein fits into a cylinder with an axial length of 238 Å and a radius of 47.5 Å, in which the eight β‐crustacyanin molecules are probably arranged in a helical manner.

Carotenoproteins: advances in structure determination

- The amino acid sequences of the two main subunits (CRTA and CRTC) of acrustacyanin. the lobster carapace carotenoprotein. have been determined. Computer graphics have been used to model the structures of the subunits, that of the dimer. D-crustacyanin, and the putative binding-sites for astaxanthin. Progress in determining the crystal structures and advances in sequencing carotenoproteins from other invertebrate phyla is reported.

Studies on a blue carotenoprotein, linckiacyanin, isolated from the starfish Linckia laevigata (Echinodermata: Asteroidea)

Abstract 1. 1. A blue carotenoprotein, linkiacyanin (λmax 395 and 612 nm), has been isolated from the skin of the starfish Linckia laevigata, together with a yellow carotenoprotein (λmax 402 nm; molecular size ca 0.5 × 103 kDa). 2. 2. Linckiacyanin has a large molecular size (> 103 kDa) with a predominant glycoprotein subunit of only 6 kDa. 3. 3. Two keto-carotenoids are present in the carotenoprotein: astaxanthin and the previously uncharacterized aromatic carotenoid, hydroxyclathriaxanthin. Zeaxanthin is also present. The minimum molecular weight estimate (16 kDa) implies at least 200 carotenoid molecules per linckiacyanin molecule. 4. 4. Linckiacyanin appears as discs (200–260 A diameter) and spring-like structures in electron micrographs. 5. 5. Variation in the E 612 395 ratio for linkiacyanin preparations and polydispersity in molecular sieving is discussed in the light of circular dichroism and other studies. 6. 6. The properties of linckiacyanin are compared with those of other echinoderm and crustacean carotenoproteins.

The structure and refinement of apocrustacyanin C2 to 1.3 Å resolution and the search for differences between this protein and the homologous apoproteins A1 and C1

The blue carotenoprotein alpha-crustacyanin of Homarus gammarus lobster carapace is comprised chemically of five 20 kDa subunits. Only two genes for the proteins have been isolated (J. B. C. Findlay, personal communication) and the five apoproteins fall into two sets of homologous proteins based on their chemical properties (CRTC, consisting of apoproteins C(1), C(2) and A(1), and CRTA, consisting of apoproteins A(2) and A(3)). The diffraction quality of apo C(2) has been improved from 2.2 to 1.3 A and its structure solved. The structure is compared with the A(1) and C(1) proteins determined at 1.4 A [Cianci et al. (2001), Acta Cryst. D57, 1219-1229] and 1.15 A, respectively [Gordon et al. (2001), Acta Cryst. D57, 1230-1237] and found to be very similar. Normalized B-factor difference plots per residue of different types were used to try to find chemically modified residues; none were found at these resolutions. It remains possible that the differences between the CRTC proteins result from differences in amidation. By comparison of a crystal grown with glycerol (studied at 1.6 A) and one grown without glycerol (studied at 1.3 A) it was seen that glycerol bound at the astaxanthin site.

Apocrustacyanin C1 crystals grown in space and on earth using vapour‐diffusion geometry: protein structure refinements and electron‐density map comparisons

Models of apocrustacyanin C(1) were refined against X-ray data recorded on Bending Magnet 14 at the ESRF to resolutions of 1.85 and 2 A from a space-grown and an earth-grown crystal, respectively, both using vapour-diffusion crystal-growth geometry. The space crystals were grown in the APCF on the NASA Space Shuttle. The microgravity crystal growth showed a cyclic nature attributed to Marangoni convection, thus reducing the benefits of the microgravity environment, as reported previously [Chayen et al. (1996), Q. Rev. Biophys. 29, 227-278]. A subsequent mosaicity evaluation, also reported previously, showed only a partial improvement in the space-grown crystals over the earth-grown crystals [Snell et al. (1997), Acta Cryst. D53, 231-239], contrary to the case for lysozyme crystals grown in space with liquid-liquid diffusion, i.e. without any major motion during growth [Snell et al. (1995), Acta Cryst. D52, 1099-1102]. In this paper, apocrustacyanin C(1) electron-density maps from the two refined models are now compared. It is concluded that the electron-density maps of the protein and the bound waters are found to be better overall for the structures of apocrustacyanin C(1) studied from the space-grown crystal compared with those from the earth-grown crystal, even though both crystals were grown using vapour-diffusion crystal-growth geometry. The improved residues are on the surface of the protein, with two involved in or nearby crystal lattice-forming interactions, thus linking an improved crystal-growth mechanism to the molecular level. The structural comparison procedures developed should themselves be valuable for evaluating crystal-growth procedures in the future.

Crystallization of apocrustacyanin on the International Microgravity Laboratory (IML-2) mission.

Rod-shaped crystals of apocrustacyanin C1 have been grown under microgravity on the International Microgravity Laboratory (IML-2) NASA space shuttle mission using the vapour-diffusion set-up of the Advanced Protein Crystallization Facility (APCF). The crystals obtained under microgravity are compared with crystals grown simultaneously in ground control experiments in identical APCF reactors, and with those obtained in the laboratory. The degree of reproducibility of the results in microgravity was also tested. Statistically, the microgravity-grown crystals are larger and of better X-ray diffraction quality than those grown in the ground controls but inferior to the best crystals grown in sitting drops, in the laboratory. Diffracting crystals, the best to 2.3 A, were produced in seven out of the eight reactors in microgravity, whereas the eight ground control reactors yielded only one poorly formed crystal suitable for diffraction studies, which also diffracted to 2.3 A. The crystals belong to the space group P2(1)2(1)2(1) with two subunits per asymmetric unit.

The quaternary structure of the lobster carapace carotenoprotein, crustacyanin: Studies using cross-linking agents

Abstract 1. 1. Three bis(imidoesters) of different span ( ca 9–11 A) have been used to form intermolecular cross-links between the apoproteins of the lobster carapace carotenoprotein, α-crustacyanin. 2. 2. Dimethylpimelimidate(DMP) is the most effective of the three reagents in cross-linking the oligomeric α-crustacyanin, giving predominantly dimers between apoproteins from each of the two apoprotein classes. The native dimers, β-crustacyanins, are effectively cross-linked with this reagent. 3. 3. The stability of DMP cross-linked α-crustacyanin and of the native carotenoprotein to urea treatment and to heating have been compared. 4. 4. Reagents of longer (sulpho- N -hydroxy-succinimide ester; 18 A) or shorter (1,5-difluoro-2,4-dinitrobenzene; 5 A) spans than the bis(imidoesters) are similarly able to form cross-linked dimers with the crustacyanins, but less effectively under the conditions of the reactions. 5. 5. The results are discussed in relation to the previously presented putative structure of β-crustacyanin (Keen et al. 1990b. Eur. J. Biochem. (submitted); Zagalsky et al. , 1990. Comp. Biochem. Physiol. 97B , 1–18) and to an alternative subunit interface arrangement of the apoproteins for the dimer.