Tandemly repeating peptide motifs and their secondary structure in Ceratitis capitata eggshell proteins Ccs36 and Ccs38.

Evidence from amino acid composition, Fourier transform analysis of primary structure and secondary structure prediction suggests a tripartite structure for Ceratitis capitata eggshell proteins Ccs36 and Ccs38, which consists of a central domain and two flanking 'arms'. The proteins, apparently, contain tandemly repeating peptide motifs specific for each domain of the tripartite structure. The central domain of both proteins, which exhibits extensive sequence homology with the corresponding domains of Drosophila melanogaster proteins s36 and s38, is formed by tandem repeats of an octapeptide-X-X-X-Z-Z-Z-Z-Z- (where X = large hydrophobic residue and Z = beta-turn former residue) and its variants. It is predicted to adopt a compact, most probably twisted, antiparallel beta-pleated sheet structure of beta-sheet strands regularly alternating with beta-turns or loops. The central domains of Ccs36 and Ccs38 share structural similarities, but they are recognizably different. The 'arms' of the proteins presumably serving for protein and species-specific functions differ substantially from those of Drosophila melanogaster. In Ccs36, the C-terminal 'arm' is formed by, almost precise, tandem repeats of an octapeptide-Y-X-A-A-P-A-A-S- (X = G or S), whereas the N-terminal 'arm' contains repeats of the octapeptide -Z-Z-Z-A-X-A-A-Z- (X = Q, N or E and Z a beta-turn former). In both 'arms' alpha-helices are predicted, alternating with beta-turns.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chorion genes of the medfly, Ceratitis capitata, I: Structural and regulatory conservation of the s36 gene relative to two Drosophila species.

We have used low stringency screening with the Drosophila melanogaster s36 chorion gene to recover its homologue from genomic and cDNA libraries of the medfly, Ceratitis capitata. The same gene has also been recovered from a genomic library of D. virilis. The medfly s36 gene shows similar developmental specificity as in Drosophila (early choriogenesis). It is also specifically amplified in ovarian follicles; this is the first report of chorion gene amplification outside the genus Drosophila. Alignments of s36 sequences from three species show that, in addition to its regulatory conservation, the s36 gene is extensively conserved in sequence, in a region corresponding to a central protein domain, and in short regions of 5' flanking DNA that might correspond to cis-regulatory elements.     

LepChorionDB,a database of Lepidopteran chorion proteins and a set of tools useful for the identification of chorion proteins in Lepidopteran proteomes

Chorion proteins of Lepidoptera have a tripartite structure, which consists of a central domain and two, more variable, flanking arms. The central domain is highly conserved and it is used for the classification of chorion proteins into two major classes, A and B. Annotated and unreviewed Lepidopteran chorion protein sequences are available in various databases. A database, named LepChorionDB, was constructed by searching 5 different protein databases using class A and B central domain-specific profile Hidden Markov Models (pHMMs), developed in this work. A total of 413 Lepidopteran chorion proteins from 9 moths and 1 butterfly species were retrieved. These data were enriched and organised in order to populate LepChorionDB, the first relational database, available on the web, containing Lepidopteran chorion proteins grouped in A and B classes. LepChorionDB may provide insights in future functional and evolutionary studies of Lepidopteran chorion proteins and thus, it will be a useful tool for the Lepidopteran scientific community and Lepidopteran genome annotators, since it also provides access to the two pHMMs developed in this work, which may be used to discriminate A and B class chorion proteins. LepChorionDB is freely available at http://bioinformatics.biol.uoa.gr/LepChorionDB.     

Purification and structural characterization of the central hydrophobic domain of oleosin

The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of beta-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the beta-sheet structure, present in the central domain, was arranged in parallel to the intermolecular beta-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable beta-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.     

Secondary structures of a new class of lipid body proteins from oilseeds.

The three main isoforms of the 19-kDa lipid body proteins (oleosin) have been purified to homogeneity from embryos of rapeseed. The secondary structures of these proteins as derived from circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy were compared with the secondary structures predicted from the primary sequences. The salient feature of the primary sequence of all oleosins is its division into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains, respectively. Using a variety of predictive methods based on primary amino acid sequence data, the oleosins exhibited a high probability of beta-strand structure in the 70-residue central hydrophobic domain, with relatively little alpha-helical content. Secondary structure data derived from CD and FTIR were consistent with the predictions from primary sequence, showing that the oleosins contained about 45% beta-strand and 13% alpha-helical structure. Under high salt conditions, a 40-kDa polypeptide was obtained from purified preparations of the 19-kDa oleosins. The 40-kDa polypeptide has a very similar secondary structure, as analyzed by CD and FTIR, to that of the 19-kDa oleosins. This polypeptide is therefore probably a dimer of the 19-kDa oleosins that is formed in high salt environments. A model of the general structure of oleosins is proposed whereby the central hydrophobic domain of the protein with a predominantly beta-strand structure is embedded into the non-aqueous phase of lipid-bodies. This hydrophobic region is flanked by putative alpha-helical structures in the polar N- and C-terminal domains which are probably oriented at the lipid-water interface.     

Hexapeptide Tandem Repeats Dictate the Formation of Silkmoth Chorion,a Natural Protective Amyloid

Silkmoth chorion is a fibrous structure composed mainly of two major protein classes, families A and B. Both families of silkmoth chorion proteins present a highly conserved, in sequence and in length, central domain, consisting of Gly-rich tandem hexapeptide repetitive segments, flanked by two more variable N-terminal and C-terminal arms. Primary studies identified silkmoth chorion as a functional protective amyloid by unveiling the amyloidogenic properties of the central domain of both protein families. In this work, we attempt to detect the principal source of amyloidogenicity of the central domain by focusing on the role of the tandem hexapeptide sequence repeats. Concurrently, we discuss a possible mechanism for the self-assembly of class A protofilaments, suggesting that the aggregation-prone hexapeptide building blocks may fold into a triangle-shaped β-helical structure.     

Structural and functional relationships in DnaK and DnaK756 heat-shock proteins from Escherichia coli.

The secondary structures of DnaK and the mutant DnaK756 heat-shock proteins from Escherichia coli have been investigated by Fourier transform infrared spectroscopy. The analysis of infrared data showed that DnaK and DnaK756 proteins have different secondary structures that are not affected by the presence of ATP or beta, gamma-methyleneadenosine 5'-triphosphate. The infrared data indicate also that the tertiary structures of DnaK and DnaK756 proteins are different and that DnaK protein undergoes conformational changes in its tertiary structure not only during binding of ATP but also during ATP hydrolysis. Using fluorescence spectroscopy of a single tryptophan located in the N-terminal domain of DnaK protein and fluorescence of 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which interacts with hydrophobic domains of DnaK protein, we were able to distinguish between two conformational states of DnaK protein. After binding of triphosphonucleotides, the C-terminal domain of DnaK protein changes in tertiary structure in such a way that fewer hydrophobic segments are exposed on the surface of the protein. After ATP hydrolysis, the number of hydrophobic segments on the surface of the protein is further reduced, and moreover the tertiary structure of the N-terminal domain of the protein changes. These data are discussed in terms of structural and functional relationships of both DnaK and DnaK756 proteins.     

Conformational study of silklike peptides modified by the addition of the calcium-binding sequence from the shell nacreous matrix protein MSI60 using 13C CP/MAS NMR spectroscopy

The calcium-binding site of the pearl oyster (Pinctada fucata) nacreous layer matrix protein MSI60 was introduced between different Ala-Gly repeating regions derived from the primary sequences of several silk fibroins. Several different organic solvents whose effect on the repetitive domains of silk peptides is well-understood were used to modify the secondary structure of the flanking Ala-Gly repeating regions. The local conformations of the flanking Ala-Gly repeating regions as well as the calcium-binding motif, MSI60, were determined by 13C CP/MAS NMR spectroscopy. The secondary structures of the polyalanine, poly(Ala), domains were modified by the solvent treatments in a predictable fashion, suggesting that only the solvent treatment and not the conformation of the MSI60 domain affected the conformation of poly(Ala) regions. Ala-Gly domains behaved differently, taking random coil conformation regardless of the choice of solvent, indicating that their secondary structure is affected by the central MSI60 domain. The conformation of the MSI60 domain is not altered by the solvent treatments, suggesting that it may retain its ability to bind calcium ions. This was confirmed using a calcium-binding assay. The assay further showed that the calcium-binding capability of MSI60 in the synthetic peptides was most effective when the flanking domain was in the beta-sheet structure.     

Structural models of the evolutionarily conservative central domain of silk-moth chorion proteins

Silk-moth chorion proteins belong to a small number of families: A, B, C, Hc-A and Hc-B. The central domain is an evolutionarily conservative region in each family, of variable length and composition between families. This domain shows clear 6-fold periodicities for various amino acid residues, e.g. glycine. The periodicities, together with the well-documented prevalence of beta-sheet and beta-turn secondary structure of chorion proteins, strongly support a structural model in which four-residue beta-strands alternate with beta-turns, forming a compact antiparallel, probably twisted beta-sheet. Conformational analysis, aided by interactive graphics refinement and recent experimental findings, further suggest that this structure consists of beta-strands, alternating with I' and II' beta-turns, and apparently forms the basis for the molecular and supramolecular assembly of chorion.     

Amino acid periodicities and their structural implications for the evolutionarily conservative central domain of some silkmoth chorion proteins

The central domain is an evolutionarily conservative region that is invariant in length in the A and Hc-A families of silkmoth chorion proteins. This domain shows strong sixfold periodicities for various amino acid residues, such as glycine and large non-polar residues. The periodicities and their phase relationships, together with the documented prevalence of beta-sheets and beta-turns in the chorion, strongly support a secondary structure model in which short (4-residue) beta-sheet strands alternate with beta-turns, forming a compact antiparallel, probably twisted beta-sheet. This structure should be important for the establishment of higher order structure in the chorion.     

Secondary structure and compliance of a predicted flexible domain in kinesin-1 necessary for cooperation of motors

Although the mechanism by which a kinesin-1 molecule moves individually along a microtubule is quite well-understood, the way that many kinesin-1 motor proteins bound to the same cargo move together along a microtubule is not. We identified a 60-amino-acid-long domain, termed Hinge 1, in kinesin-1 from Drosophila melanogaster that is located between the coiled coils of the neck and stalk domains. Its deletion reduces microtubule gliding speed in multiple-motor assays but not single-motor assays. Hinge 1 thus facilitates the cooperation of motors by preventing them from impeding each other. We addressed the structural basis for this phenomenon. Video-microscopy of single microtubule-bound full-length motors reveals the sporadic occurrence of high-compliance states alternating with longer-lived, low-compliance states. The deletion of Hinge 1 abolishes transitions to the high-compliance state. Based on Fourier transform infrared, circular dichroism, and fluorescence spectroscopy of Hinge 1 peptides, we propose that low-compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas high-compliance states correspond to the loss of that structure. We hypothesize that strain accumulated during multiple-kinesin motility populates the high-compliance state by unfolding helical secondary structure in the central Hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiple-motor situations.     

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.     

Structural features of B family chorion sequences in the silkmoth Bombyx mori, and their evolutionary implications.

Partial protein sequences, and DNA sequences of corresponding cDNA and genomic clones were obtained and analyzed to reveal the primary structural features of major, developmentally middle or late components of the B chorion multigene family in Bombyx mori. Comparisons with other types of sequences confirm and clarify the tripartite domain structure of chorion proteins. Glycine-, leucine- and tyrosine-containing, tandemly repetitive peptides form the bulk of the amino-terminal and carboxy-terminal domains ('arms'). Extensive sequence homologies suggest a common evolutionary origin for the amino-terminal arms of some B. mori B sequences and the corresponding portions of members of a different (A) chorion multigene family in Antheraea polyphemus, a distantly related silkmoth.     

Identification and time of synthesis of chorion proteins in Drosophila melanogaster.

The chorion of Drosophila melanogaster consists of proteins secreted by the follicular epithelium during late oogenesis. Petri, Wyman and Kafatos (1976) have described six major protein components of the Drosophila chorion and reported the synthesis of these proteins in vitro by mass-isolated egg chambers. We have used two-dimensional gel electrophoresis to identify approximately twenty components in highly purified chorion preparations. The synthesis patterns of these proteins in vivo were determined by isolating egg chambers of different developmental stages from flies injected with 14C amino acids. Chorion proteins constitute a large fraction of the protein synthesized by ovarian egg chambers in stages 12--14. The sizes and times of synthesis of the chorion proteins correlate closely with the production of poly(A)-containing RNAs by the follicle cells (Spradling and Mahowald, 1979).