Laser-Raman and infrared spectroscopic studies of protein conformation in the eggshell of the fish Salmo gairdneri

Laser-Raman and infrared spectroscopic studies reveal abundant beta-pleated sheet conformation in the eggshell proteins of the fish Salmo gairdneri. This secondary structure is the underlying molecular conformation, dictating the formation of the helicoidal architecture of the eggshell. Disulphide bonds crosslink the eggshell proteins of the fertilized eggs and are apparently found in g-g-g (gauche-gauche-gauche), g-g-t (gauche-gauche-trans) and t-g-t (trans-gauche-trans) conformation. There is no evidence for the existence of free sulphydryls. The tyrosines appear to act as hydrogen-bond acceptors, whereas the aromatic residues phenylalanine and tryptophan are also eggshell protein constituents.     

Twisted β-pleated sheet: the molecular conformation which possibly dictates the formation of the helicoidal architecture of several proteinaceous eggshells

Evidence from X-ray diffraction, laser-Raman spectroscopy, secondary structure prediction, freeze-fracturing, conventional electron microscopy and Fourier analysis suggests that the helicoidal structure of the silkmoth eggshell (chorion) is created by protein molecules, most probably in a twisted β-pleated sheet conformation. It is proposed that this conformation also dictates the formation of the helicoidal architecture of other proteinaceous eggshells; apparently, it may also play an important role in the formation of the helicoidal architecture in other biological systems with protein components.     

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro

We investigated matrix–mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix–mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {1 0 4} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {1 0 4} calcite faces. OPN inhibited calcite growth in vitro at the {1 0 4} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.     

Helicoidal architecture of fish eggshell

Previous publications show arced patterns in electron micrographs of either microfibrils or canals in sectioned fish eggshells, but these have been misinterpreted. We show here that such patterns in the inner layer of cod (Gadus morrhua), plaice (Pleuronectes platessa) and trout (Salmo gairdneri) eggs arise from a helicoidal structure. This consists of a laminate of protein microfibrils, with the direction of ply processing like the steps of a spiral staircase and with the same sense as a left-handed corkscrew. Mechanically, this is an ideal way to strengthen a spherical shell, to resist deforming forces equally from any direction. Radial canals which traverse this layer are forced into flattened and twisted ribbons. Both the helicoidal microfibrillar structure and the canal shape in fish eggshells show remarkable convergent evolution with similar structures in insect cuticles. Trout eggs were resistant to deforming forces as high as 380,000 N/m². Fish eggshells, like those of many other organisms, are mechanically well designed.     

The trichurid egg-shell: Evidence in support of the Bouligand hypothesis of helicoidal architecture

Electron microscopy of thin sections and freeze etch replicas of the eggs of the nematodes Trichuris suis and T. muris is used to provide evidence in support of the Bouligand hypothesis of helicoidal architecture. The evidence presented is as follows:

1. 1. The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt.

2. 2. Sections cut normal to the plane of the laminae do not show parabolic patterning. Parabolae appear if the section is tilted—their direction depending upon the direction of tilting.

3. 3. Freeze etching allows the direct visualization of helicoidal architecture. Fibres are parallel within any one lamina but the fibre direction rotates by an angle of 9 ° in successive laminae. Parabolic arcs are made up of short lengths of straight fibres—curved fibres were not observed.

Planes of sectioning producing single and double spiral artifacts are described and the formation of these artifacts discussed. The sense of rotation of the helicoid is shown to be asymmetrical about any mid-plane through the egg.     

Secondary structure of chorion proteins of the Lepidoptera Pericallia ricini and Ariadne merione by ATR FT-IR and micro-Raman spectroscopy

The gross morphological features of the eggs and eggshells (chorions) of two Lepidoptera species, Pericallia ricini and Ariadne merione were revealed for the first time by scanning and transmission electron microscopy. These two insect pests are extremely serious threats for many crops, mainly in India, but also in several other regions of the world. Micro-Raman and ATR FT-IR spectroscopy were also applied to study in detail the secondary structure of the eggshell (chorion) proteins of these Lepidoptera species. Both techniques indicate that the two species have nearly identical conformations of their chorion proteins with abundant antiparallel β-pleated sheet. These results are in support of our previous findings that the helicoidal architecture of the proteinaceous chorion of Lepidoptera and fishes is dictated by a common molecular denominator, the antiparallel β-pleated sheet secondary structure.     

Cracks in the shell—zooming in on eggshell formation in the human parasite <Emphasis Type="Italic">Schistosoma mansoni</Emphasis>

Schistosomiasis, currently the second most common parasitic disease of humans in tropical regions is caused by the eggs of trematode worms of the genus Schistosoma. Understanding egg formation and specifically the synthesis of the eggshell comprises, consequently, a promising starting point to cure and prevent the disease. To shed light on the genetics of the latter process, we analysed the three known S. mansoni eggshell proteins P14, P19 and P48 against the background of the species inferred proteome and of eggshell proteins identified in other trematode species. Our results suggest that eggshell formation in Schistosoma involves a multitude of different proteins organised in currently three distinct protein families (P14, P48 and P34 eggshell protein family). The first two families are of simple structure. Their respective members share a substantial degree of sequence similarity and are, to date, observed only in the genus Schistosoma. In contrast, the P34 family of eggshell proteins is complex. Its in part highly diverged members share only a conserved motif of 67-aa length on average and are detected in various trematode species. The resulting widespread occurrence of this protein motif suggests an important role during eggshell formation in trematodes. Screening more than 7,000 putative proteins of S. mansoni, we could identify six new members of the P34 protein family that are likely to be involved in eggshell formation in this species.Electronic Supplementary Material Supplementary material is available for this article at .     

Laser-Raman spectroscopic studies of the eggshell (chorion) of Bombyx mori

Laser-Raman spectroscopic studies of the eggshell (chorion) of the silkmoth Bombyx mori reveal that its component proteins consist of 60–70% antiparallel β-pleated sheet and 30–40% of β-turns. The disulphide bonds, which crosslink the (extremely rich in cysteine)-proteins of the outer lamellar eggshell layer, are apparently found in G-G-G (gauche-gauche-gauche) and T-G-T (trans-gauche-trans) conformation; there is no evidence for the existence of free sulphydryls. The highly localized tyrosine residues appear to form hydrogen bonds, acting as weak proton donors or as acceptors.     

Drosophila vitelline membrane assembly: A critical role for an evolutionarily conserved cysteine in the “VM domain” of sV23

The vitelline membrane (VM), the oocyte proximal layer of the Drosophila eggshell, contains four major proteins (VMPs) that possess a highly conserved “VM domain” which includes three precisely spaced, evolutionarily conserved, cysteines (CX⁷CX⁸C). Focusing on sV23, this study showed that the three cysteines are not functionally equivalent. While substitution mutations at the first (C123S) or third (C140S) cysteines were tolerated, females with a substitution at the second position (C131S) were sterile. Fractionation studies showed that sV23 incorporates into a large disulfide linked network well after its secretion ceases, suggesting that post-depositional mechanisms are in place to restrict disulfide bond formation until late oogenesis, when the oocyte no longer experiences large volume increases. Affinity chromatography utilizing histidine tagged sV23 alleles revealed small sV23 disulfide linked complexes during the early stages of eggshell formation that included other VMPs, namely sV17 and Vml. The early presence but late loss of these associations in an sV23 double cysteine mutant suggests that reorganization of disulfide bonds may underlie the regulated growth of disulfide linked networks in the vitelline membrane. Found within the context of a putative thioredoxin active site (CXXS) C131, the critical cysteine in sV23, may play an important enzymatic role in isomerizing intermolecular disulfide bonds during eggshell assembly.     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.     

Prediction ofcis/transisomerization in proteins using PSI-BLAST profiles and secondary structure information

Background  

The majority of peptide bonds in proteins are found to occur in thetransconformation. However, for proline residues, a considerable fraction of Prolyl peptide bonds adopt thecisform. Prolinecis/transisomerization is known to play a critical role in protein folding, splicing, cell signaling and transmembrane active transport. Accurate prediction of prolinecis/transisomerization in proteins would have many important applications towards the understanding of protein structure and function.     

cuticleDB: a relational database of Arthropod cuticular proteins

Background  

The insect exoskeleton or cuticle is a bi-partite composite of proteins and chitin that provides protective, skeletal and structural functions. Little information is available about the molecular structure of this important complex that exhibits a helicoidal architecture. Scores of sequences of cuticular proteins have been obtained from direct protein sequencing, from cDNAs, and from genomic analyses.     

Molecular Dynamics Simulation Study of DNA Triplex Formed by Mixed Sequences in Solution

Abstract

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC)?d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usual “cut-off” of the long—range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence—dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other legends.     

The eggshell is required for meiotic fidelity, polar-body extrusion and polarization of the C. elegans embryo

Background  

Fertilization restores the diploid state and begins the process by which the single-cell oocyte is converted into a polarized, multicellular organism. In the nematode, Caenorhabditis elegans, two of the earliest events following fertilization are secretion of the chitinous eggshell and completion of meiosis, and in this report we demonstrate that the eggshell is essential for multiple developmental events at the one-cell stage.     

A new model for cellulose architecture in some plant cell walls

Summary A new model of rotating fibre components (helicoidal model) is proposed to explain the architecture of some plant cell walls. On the basis of tilting observations under the electron microscope, we establish the validity of this model for the cell wall ofChara vulgaris oospores. We suggest that this model explains the architecture seen in a number of published micrographs from a variety of different plant cell walls. Helicoidal architecture is shown to be distinct from the previously established crossed polylamellate architecture. The diagnostic features of helicoidal architecture are given. Morphogenesis of plant cell walls is discussed, with particular reference to self assembly in cholesteric liquid crystals.     

Genome-wide association study revealed a promising region and candidate genes for eggshell quality in an F2 resource population

Background

Eggshell is subject to quality loss with aging process of laying hens, and damaged eggshells result in economic losses of eggs. However, the genetic architecture underlying the dynamic eggshell quality remains elusive. Here, we measured eggshell quality traits, including eggshell weight (ESW), eggshell thickness (EST) and eggshell strength (ESS) at 11 time points from onset of laying to 72 weeks of age and conducted comprehensive genome-wide association studies (GWAS) in 1534 F₂ hens derived from reciprocal crosses between White Leghorn (WL) and Dongxiang chickens (DX).

Results

ESWs at all ages exhibited moderate SNP-based heritability estimates (0.30 ~ 0.46), while the estimates for EST (0.21 ~ 0.31) and ESS (0.20 ~ 0.27) were relatively low. Eleven independent univariate genome-wide screens for each trait totally identified 1059, 1026 and 1356 significant associations with ESW, EST and ESS, respectively. Most significant loci were in a region spanning from 57.3 to 71.4 Mb of chromosome 1 (GGA1), which together account for 8.4 ~ 16.5 % of the phenotypic variance for ESW from 32 to 72 weeks of age, 4.1 ~ 6.9 % and 2.95 ~ 16.1 % for EST and ESS from 40 to 72 weeks of age. According to linkage disequilibrium (LD) and conditional analysis, the significant SNPs in this region were in extremely strong linkage disequilibrium status. Ultimately, two missense SNPs in GGA1 and one in GGA4 were considered as promising loci on three independent genes including ITPR2, PIK3C2G, and NCAPG. The homozygotes of advantageously effective alleles on PIK3C2G and ITPR2 possessed the best eggshell quality and could partly counteract the negative effect of aging process. NCAPG had certain effect on eggshell quality for young hens.

Conclusions

Identification of the promising region as well as potential candidate genes will greatly advance our understanding of the genetic basis underlying dynamic eggshell quality and has the practical significance in breeding program for the improvement of eggshell quality, especially at the later part of laying cycle.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1795-7) contains supplementary material, which is available to authorized users.     

Twisting of glycosidic bonds by hydrolases

Patterns of scissile bond twisting have been found in crystal structures of glycoside hydrolases (GHs) that are complexed with substrates and inhibitors. To estimate the increased potential energy in the substrates that results from this twisting, we have plotted torsion angles for the scissile bonds on hybrid Quantum Mechanics::Molecular Mechanics energy surfaces. Eight such maps were constructed, including one for α-maltose and three for different forms of methyl α-acarviosinide to provide energies for twisting of α-(1,4) glycosidic bonds. Maps were also made for β-thiocellobiose and for three β-cellobiose conformers having different glycon ring shapes to model distortions of β-(1,4) glycosidic bonds. Different GH families twist scissile glycosidic bonds differently, increasing their potential energies from 0.5 to 9.5 kcal/mol. In general, the direction of twisting of the glycosidic bond away from the conformation of lowest intramolecular energy correlates with the position (syn or anti) of the proton donor with respect to the glycon’s ring oxygen atom. This correlation suggests that glycosidic bond distortion is important for the optimal orientation of one of the glycosidic oxygen lone pairs toward the enzyme’s proton donor.     

Evolution of thedec-1 eggshell locus inDrosophila. II. Intraspecific DNA sequence analysis reveals length mutations in a repetitive region inD. melanogaster

Summary Thedec-1 eggshell gene inDrosophila melanogaster encodes follicle cell proteins required for proper eggshell assembly. As shown by Southern and Northern analyses thedec-1 gene occurs in four alleles (Fcl-4) among wild-type strains. Its second exon has a distinct feature in the form of 12 repeats with 78–91 nucleotides; the first five show nearly 100% homology. DNA sequence comparison of the repeated region of the alleles revealed that the length polymorphisms are caused by changes in the numbers of the first five repeats. The results suggest that the alleles have been generated by unequal intragenic crossing-over and/or slippage during DNA replication and that the allelic length variants have arisen independently. The possiblilty that the most common allele,FC1, has a selective advantage over the other alleles is discussed.     

Ultrastructure and elemental composition of the eggshell of Reeve’s pheasant ( Syrmaticus reevesii )

The eggshell of Reeve’s pheasant (Syrmaticus reevesii) collected from the Dongzhai National Nature Reserve in Henan Province, China was studied. By using scanning electron microscopy and inductively coupled plasma (ICP) spectrometry, the ultrastructure and elemental composition of the eggshell was determined. The study showed that the average thickness of the surface layer of crystals, the palisade and cone layer, and the eggshell membrane were 20.8, 220.8 and 62.5 μm, respectively, accounting for 6.8%, 72.6% and 20.6% of the total thickness of the eggshell. There were many vesicular holes in the palisade layer with an average diameter of 0.32 ± 0.08 μm (n = 30). The function of these holes might be significant to air exchange. The shape of the eggshell pore on the surface layer of crystals is round or elliptical. The fracture surface of the pore is funnel-shaped. Some granules filled the upper part of the eggshell pores. The content of 21 elements in the eggshell of wild and captive Reeve’s pheasants was compared and presented. It indicated that among the elements that made up the eggshell of the wild pheasant, the content of Ca, Mg, P and S was much higher, ω > 1 mg/g, with ω (Ca) being higher than 40% of the eggshell. The contents of Na, Si, Sr, K and Al were ω = 0.1–1 mg/g, while Fe, Zn, Pb, Mn, Cu, V and Ti had lower concentrations (ω = 1–100 μg/g). The ω of Ni, Cr, Co, Se, Cd were lower than 1 μg/g. The elemental composition in the eggshell of the captive Reeve’s pheasant kept in the Dongzhai National Natural Reserve was significantly different from that of the wild species, with a difference of over 20% on S, Cu, Fe, Al, Mn, Si, Sr, Se and Cr. The lower intake of Fe, Mn, Si and Sr on the one hand and the higher intake of S, Cu, Al and Cr on the other hand might be responsible for the low fertility of captive Reeve’s pheasants in the Dongzhai National Nature Reserve. In order to ensure that the pheasants are receiving the proper amount of nutrition and to improve their breeding success, the amount of certain elements in the food should be adjusted. Translated from Journal of Beijing Normal University (Natural Science), 2006, 42(1): 78–82 [译自: 北京师范大学学报 (自然科学版)]     

Crystalline conformation of homo- and regular heteroglucan chains

Crystalline polysaccharides with 1 → 3−β and 1 → 4−β glycosidic linkages are the most prevalent ones in nature. An interpretation of recent X-ray data on 1 → 3−α glucan shows that it has a ribbon-like crystalline conformation similar to cellulose. Comparison of the crystalline conformation of the four principal homoglucans shows that they fall either in the ‘ribbon-like’ or ‘large amplitude’ helix class. Heteroglucans with a regular sequence of glucosidic linkages show characteristics of the ‘extended conformation’ rather than the ‘coiled conformation’ even when there is 50% of a linkage which in a homoglucan leads to a large amplitude helix. It is concluded that X-ray diffraction analysis fully establishes the hypothesis that the glycosidic linkage type is the determinant of polysaccharide conformation. In this respect, polysaccharides are more like synthetic polymers than proteins or nucleotides; in the latter, it is variation in the substituents which are responsible for the conformational diversity.