Deriving the ultrastructure of α-crustacyanin using lower-resolution structural and biophysical methods

The structure of α-crustacyanin has been determined to 30 Å resolution using negative-stain electron microscopy (EM) single-particle averaging and modelling with the β-crustacyanin dimer from the crystal structure (Protein Data Bank code 1gka), guided by PISA protein subunit interface calculations for 1gka, and compared with the protein arrangements observed in the crystal lattice of 1gka. This α-crustacyanin EM model has been checked against SAXS experimental data, including comparison with rigid-body models calculated from the SAXS data, and finally with analytical ultracentrifugation measurements

The association of carotenoids with protein in certain invertebrates

Crustacyanin, the blue carotenoprotein of the lobster carapace was obtained in a state homogeneous in cellulose acetate electrophoresis. It was crystallised and shown to be a globulin of large particle size, free from lipid and carbohydrate. The minimum molecular weight of the protein calculated on the carotenoid content, was found to be one-tenth of that expected from its size. Reversible changes in spectrum occurring on removal of salt were shown to be attended by dissociation into subunit of the size expected from the minimum molecular weight. Apocrustacyanin was shown to be of the subunit size and heterogeneous in cellulose acetate electrophoresis. Native cnustacyanin was reconstituted from the apoprotein and astaxanthin. Possible modes of binding of the carotenoid are suggested to account for the spectral changes occurring in urea. Electrophoretic similarities between crustacyanin treated with urea and apocrustacyanin are discussed in relation to the number of different subunits. It is suggested that crustacyanin is composed of a specific geometrical association of several small subunits, possibly different, and that the carotenoid stabilises the subunits in a configuration essential for the association and possibly also assisting in the binding. Ovoverdin, the green carotenoprotain of lobster ovary, was obtained homogeneous in cellulose acetate electrophoresis and shown to be a glycolipoprotein, precipitating at low ionic strength out soluble in distilled water. The possibility of two molecules of carotenoid being associated with each molecule of protein has been investigated. Astaxanthin-proteins were purified from the carapaces of Eriphia spinifrons, Carcinus maenas, Astacus astacus and Palinurus vulgaris. Their absorption spectra and properties arc compared with those of crustacyanin and ovoverdin. Glycolipoproteins containing a number of carotenoids have been purified from the ovaries of Carcinus maenas, Pecten maximus and Palinurus vulgaris, and from the eggs of Palinurns vulgaris. Their properties have been investigated and the ode of association of the carotenoids is discussed.<p

Development and application of novel crystallisation methodology to medically relevant proteins

X-ray crystallography is the most widely used technique for determining 3-D protein structures. Information gained from crystallography is vital to the success of rational drug design and other biotechnological applications. Crystallography relies on the production of high quality protein crystals but obtaining such crystals is a major obstacle to progress. This project tackles this problem by (i) designing, developing and validating new and improved crystallisation methodologies for obtaining high quality crystals; and (ii) applying the new techniques in addition to existing methods, to crystallise medically important proteins in order to facilitate their 3-Dimensional structure determination. Presented here is the design and validation of an improved method to slow down protein crystallisation in order to enable the growth of fewer, larger single crystals of higher quality. The method called the oil-on-drop method is a variation of the Chayen method which places an oil barrier over precipitant reservoirs in hanging drop trials. The variation consists of dispensing the oil directly onto the protein drops. This method was successfully tested and validated using five proteins, three of which are of medical relevance including a methyltransferase and an antibody-peptide complex. A comparison of the oil-on-drop method with existing standard and non-standard techniques for optimising crystal quality such as the application of nucleants and the Chayen method showed the oil-on-drop technique to be superior in many ways. Most importantly, it reproducibly yielded crystals diffracting to higher resolution of three of the proteins tested namely the methyltransferase, the complex and trypsin. In the case of the Roab13 antibody-peptide complex the crystals obtained were of the highest resolution than ever attained previously. In addition, the oil-on-drop method overcomes problems encountered by other methods. It can be used with a wider variety of precipitating agents and can be performed using robots in a high throughput mode.Open Acces

A review of carotenoid utilisation and function in crustacean aquaculture

Crustaceans are cultured extensively around the world in intensive farming systems. High-performance formulated feeds have been developed for crustacean aquaculture, which are often supplemented with a number of natural and synthetic carotenoid sources. Studies over a number of years have consistently shown that dietary carotenoid supplementation is beneficial for crustacean aquaculture across a range of commercially relevant parameters. Most obvious is the effect on pigmentation, where carotenoid inclusion levels in feeds and duration of feeding diets with carotenoids have been optimised across many species to improve product colour, and subsequently quality and price. However, beneficial effects of carotenoid inclusion have increasingly been demonstrated on other parameters. This review updates the recent progress in our understanding of dietary carotenoid utilisation and storage, and the combined effects of diet, genetics and environment on crustacean pigmentation. In addition, the range of other physiological benefits this class of molecules brings to these animals is summarised. These include improvements in survival, growth, reproductive capacity, disease resistance and stress resistance. &copy; 2015 Wiley Publishing Asia Pty Ltd

Extraction, Purification and partial Characterization of a Carotenoid Binding Protein (CBP) from the Epidermis of the Monarch Butterfly Larvae (Danaus plexippus)

This dissertation describes the purification and partial characterization of CBP from the epidermis of the monarch butterfly larvae (Danaus plexippus). A yellow protein-carotenoid complex was extracted from the yellow pigmented epidermal tissue from monarch butterfly larvae by homogenization. Additional steps in the purification process included differential precipitation with ammonium sulfate, cation and anion chromatography, and lastly size exclusion chromatography. Polyacrylamide gel electrophoresis demonstrates that a single protein was isolated (M-LBP) having a ~60 kDa molecular weight, the value has subsequently been confirmed by HR-tandem MS. Lutein is the sole carotenoid bound by M-LBP with a stoichiometry of the binding of 2: 1. Immunohistochemistry results show that M-LBP has no cross-reactivity to antibodies for silk worm CBP (Bombix mori) but does have cross-reactivity with antibodies for horn worm epidermal CBP (Agrius convolvuli). Binding affinities were determined using surface plasmon resonance for the carotenoids lutein (KD = 18.6 ± 0.7), R,R-zeaxanthin (KD = 990 ± 60), R,S-zeaxanthin (KD = 60 ± 2). Tryptophyphan fluorescence lifetimes were determined for the apoprotein and compared to those of the native M-LBP. Tryptophan fluorescence lifetimes were found to be 3.9 ns and 3.0 ns, respectively for these two forms of the protein, indicating that upon dissociation of the carotenoid from the protein the tryptophan fluorophore adopts a position where it has less interaction with the polar surface environment

Transcriptome analysis of <em>Litopenaeus vannamei</em> during the early stage of limb regeneration process

Regeneration is a process in which organisms regrow new tissues or organs at the injury site, which has attracted the attention of many scientists and nonscientists. However, the underlying molecular mechanisms of regeneration after autotomy are largely unknown. In this study, we conducted RNA-seq sequencing on regenerated limb bud tissues of *Litopenaeus vannamei* at 0 hours post autotomy (0 hpa), 12 hours post autotomy (12 hpa), and 24 hours post autotomy (24 hpa). A total of 2,192 differentially expressed genes related to energy metabolism, transcription and translation, and epidermis development were identified between 0 hpa and 12 hpa, such as triosephosphate isomerase A, triosephosphate isomerase B, and zinc finger protein 367 that is upregulated in 12 hpa. Between 12 hpa and 24 hpa, 1,447 differentially expressed genes were identified that were related to cuticle development and energy metabolism, such as cuticle protein 6, which is upregulated in 24 hpa, and triosephosphate isomerase is downregulated in 24 hpa. The results indicated that energy metabolism, transcription and translation, epidermal formation, and chitin metabolism processes are involved during the early stage of limb regeneration. This study provides basic knowledge for investigating the molecular mechanisms associated with limb regeneration in crustaceans at the early regeneration stage

An hypothesis on crustacean pigmentation metabolism:L-carnitine and nuclear hormone receptors as limiting factors

Astaxanthin (Axn) is the primary pigment molecule in crustaceans associated with quality, health and growth traits, leading to increased marketing value. Axn can be contained within the protein complex crustacyanin (CRCN) to produce an array of different shell colours, or esterified with fatty acids (FA) for storage but also contributing additional red colouration. l-Carnitine (LC) has a major role in FA oxidation and mitochondrial function optimization, which could influence the proportion of Axn complexed with FA or CRCN. Peroxisome proliferator activated receptors (PPARs) have important roles in FA and Axn uptake, and stored lipid oxidation affecting Axn homeostasis and storage in lipid bodies. Whether Axn could increase PPAR signalling and carnitine palmitoyl transferase activity, leading to induction of lipid metabolism, is not known in crustaceans. Several FA have been shown to preferentially form FA Axn-esters, including saturated fatty acids (SFA) such as C16:0 and C18:0, mono-unsaturated fatty acids (MUFA) such as C16:1 and C18:1, and poly-unsaturated fatty acids (PUFA) such as C20:4, C20:5, and C20:6. We hypothesize that manipulating the dietary ratios and inclusion of LC, Axn, and specific FA may be able to further improve pigment utilization, lipid metabolism, health, and growth in crustaceans.</p

Circular dichroism study of the light-harvesting PCP complexes within the vibronic multistate dimer theory

Chiral properties of peridinin-chlorophyll-protein (PCP) light-harvesting complexes are studied in terms of vibronic dimer theory previously applied to study certain structural aspects of α-crustacyanin pigments. On the base of CD spectra it is shown that the peridinin dimer acts as a chiral group in PCP complexes and its geometrical structure is such that the peridinin monomers cannot be coplanar. Certain observations concerning the energy transfer process in PCP complexes in vivo are also made