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...

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.

A comprehensive study of microgravity and ground-grown chicken egg-white lysozyme crystals is presented using synchrotron X-ray reciprocal-space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed reduced intrinsic mosaicities on average, but no differences in terms of strain over their ground-grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the ground-control case only a small volume of the crystal contributed to the intensity at the diffraction peak. The techniques prove to be highly complementary, with the reciprocal-space mapping providing a quantitative measure of the crystal mosaicity and strain (or variation in lattice spacing) and the topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out at the synchrotron.

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The drive for yet higher output of protein crystal structures sustains an interest in streamlining data-collection protocols. Highly organized automated robotics attend to one aspect. The data-collection elapsed time also depends on the number of wavelengths used, the data redundancy and the desired diffraction resolution limit. Synchrotron-radiation beamlines offer a centralized data-collection approach with considerable flexibility of choice of wavelength(s) with high beam intensity and fine collimation. The arrival of automated IPs (imaging plates) and CCDs (charge-coupled devices) with sample freezing has widened the scope for ease of data collection and achieving a high redundancy, the latter at the cost of elapsed time per sample. This paper reports a single-wavelength approach where a large optimized f" signal involving the Xe L1-absorption edge is harnessed. Even with modest data redundancy (7), a high-quality electron-density map was obtained from that single data set when combined with phase improvement methods. Two phasing protocols are compared for the same data set. Resolution extension (to 1.4 A), in this test, was also done but via a second data set. In future, with a tilted detector geometry, full diffraction resolution will be collectable in one experiment on the one beamline. Moreover, careful minimization of beamline Be window thickness on the new Daresbury SRS Multipole Wiggler Beamline 10 will maximize the 2-3 A (softer) wavelength intensity performance specification. The work has application to the harnessing of iodine f" optimized signals and enhanced sulfur f", as well as xenon f", in protein crystallography. This approach, softer-SWAT, should 'swat' many protein structures when it comes online.

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The synthesis of new bischromone derivatives (4a-c and 5a-c) as potential anticancer drugs is described. The difference in the reactivity between 4-oxo-4H-chromene-3-carboxylic acid 2 (or its methyl ester 3) and 4-oxo-4H-chromene-3-carbonyl chloride 1 with three different polyamines: 3,3'-diamino-N-methyldipropylamine (a), 1,4-bis(3-aminopropyl)piperazine (b), 4,9-dioxa-1,12-dodecanediamine (c) resulted in the formation of two different groups of products, compounds 4a-c and 5a-c, designed in agreement with the bisintercalators' structural requirements. The transformation of 4-oxo-4H-chromene-3-carboxylic acid into 2H-chromene-2,4(3H)-diones (5) was confirmed by the NMR and XRD experiments. Compounds 4a and 5a were evaluated in vitro in the highly aggressive melanoma cell line A375. An enhanced induction of apoptosis and cell cycle arrest clearly revealed that compound 5a was more potent than 4a. Compound 5a was also more active in diminishing the adhesive potential of melanoma cells. Current studies support the notion that small changes in the three-dimensional structure of molecules might have a substantial impact on their biological activity.

The A1 subunit of the carotenoprotein alpha-crustacyanin, isolated from lobster carapace, has been crystallized using the vapour-diffusion method. The crystals, grown in solutions of ammonium sulfate containing methylpentanediol (MPD), diffracted to 2. 0 A. The crystals are stable to radiation. The space group of the crystals is P2(1)2(1)2(1). The unit-cell parameters are a = 41.9, b = 80.7, c = 110.8 A. 'Standard structure determination' has been unsuccessful within this crustacyanin family. Instead, an approach based on the S atoms is being undertaken involving softer X-rays at the SRS, Daresbury.