On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences.

The repetitive sequence GGLGY was found in lamprin, the most important matrix protein of lamprey annular cartilage by Keeley and co-workers. Similar sequences appear also in other proteins, i.e. elastin, spidroin, spider minor ampullate silk proteins, in matrix proteins of the chorion or egg shell membrane of insects and others. We synthesized (GGLGY)n, n=1, 2, 6, because the sequence is repeated six times in the aggregated protein. The peptides were studied both in solution and in the solid state. Because the CD spectra were dominated by aromatic contribution, we synthesized GGLGF and GGLGA in order to carefully interpret the CD spectra. The conformational analysis suggests that all synthetic peptides do adopt the same secondary structure. In solution the peptides present a flexible conformation with a significant amount of PPII structure. In the solid state PPII, beta-pleated-sheets and beta-turns possibly co-exist.     

Polyproline II helix is a key structural motif of the elastic PEVK segment of titin

Titin is a family of giant elastic proteins that constitute an elastic sarcomere matrix in striated muscle. In the I-band region of the sarcomere, where titin extends and develops passive force upon stretch, titin is composed of tandem repeats of approximately 100 residue immunoglobin domains and approximately 28-residue PEVK modules. We have performed 2D NMR and circular dichroism (CD) studies of the conformations of one representative 28-mer PEVK module from human fetal titin (PEPPKEVVPEKKAPVAPPKKPEVPPVKV). NMR data of synthetic peptides of this module as well as three constituent peptides of 9 to 12 residues in aqueous solutions reveal distinguishing features for left-handed three-residue per turn PPII helices: the lack of NOE NN(i, i+1), very large NOE alphaN(i, i+1)/NN(i, i+1), no medium range NOE alphaN(i, i+2), and dihedral angles phi and psi values of -78 and 146, respectively. Structural determinations indicate the presence of three short stretches of PPII helices of 4, 5, and 6 residues that are interposed with an unordered, and presumably flexible, spacer region to give one "polyproline II helix-coil" or "PhC" motif for roughly every 10 residues. These peptides also display the characteristic PPII CD spectra: positive peak or negative shoulder band at 223 nm, negative CD band near 200 nm, and biphasic thermal titration curves that reflect varied stability of these PPII helices. We propose that this PhC motif is a fundamental feature and that the number, length, stability, and distribution of PPII is important in the understanding of the elasticity and protein interactions of the PEVK region of titin.     

A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.     

Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3)

Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.     

Plasmodium falciparum: elicitation by peptides and recombinant circumsporozoite proteins of circulating mouse antibodies inhibiting sporozoite invasion of hepatoma cells

The immunodominant epitope region of the circumsporozoite protein of Plasmodium falciparum sporozoites contains 37 tandem repeats of the tetrapeptide Asn-Ala-Asn-Pro and 4 repeats of Asn-Val-Asp-Pro. Synthetic peptides and recombinant proteins of the repeat region were used to immunize mice using different doses and adjuvants. Antisera were tested for inhibition of sporozoite invasion of cultured human hepatoma cells. Synthetic peptides and recombinant proteins elicited high levels of antibodies that inhibited sporozoite invasion when emulsified with complete Freund's adjuvant. Since recombinant proteins with alum elicited a better antibody response to sporozoite invasion than they did without adjuvant, it may be that a recombinant protein containing 32 tandem copies of the tetrapeptide repeat combined with alum could be a candidate malarial vaccine suitable for human trials.     

Amyloid character of self-assembling proteins based on adenovirus fiber shaft sequences

Fibrous proteins found in natural materials such as silk fibroins, spider silks, and viral spikes increasingly serve as a source of inspiration for the design of novel, artificial fibrous materials. The fiber protein from the adenovirus has previously served as a model for the design of artificial, self-assembling fibers. The fibrous shaft of this protein consists of 15-amino-acid sequence repeats that fold into a triple β-spiral motif in their native context. Recombinant proteins based on multimers of simplified consensus shaft repeats were previously reported to form self-assembling fibrils from which filaments could be spun. Here, we describe the structural characterization of these fibrils; X-ray fiber diffraction, Raman spectroscopy, and Congo Red binding strongly suggest an amyloid-type structure for these fibrils, with β-strands arranged perpendicular to the fibril axis. This amyloid structure is distinct from the native β-spiral fold, and similar to amyloid structures formed by short, synthetic peptides corresponding to shaft sequences. We discuss implications for the rational design of novel fibrous materials, based on crystal structure information and knowledge of folding and assembly pathways of natural fibrous proteins.     

The impact of either 4-R-hydroxyproline or 4-R-fluoroproline on the conformation and SH3m-cort binding of HPK1 proline-rich peptide

SH3 domains are probably the most abundant molecular-recognition modules of the proteome. A common feature of these domains is their interaction with ligand proteins containing Pro-rich sequences. Crystal and NMR structures of SH3 domains complexes with Pro-rich peptides show that the peptide ligands are bound over a range of up to seven residues in a PPII helix conformation. Short proline-rich peptides usually adopt little or no ordered secondary structure before binding interactions, and consequently their association with the SH3 domain is characterized by unfavorable binding entropy due to a loss of rotational freedom on forming the PPII helix. With the aim to stabilize the PPII helix conformation into the proline-rich decapeptide PPPLPPKPKF (P2), we replaced some proline residues either with the 4(R)-4-fluoro-l-proline (FPro) or the 4(R)-4-hydroxy-l-proline (Hyp). The interactions of P2 analogues with the SH3 domain of cortactin (SH3_m-cort) were analyzed by circular dichroism spectroscopy, while CD thermal transition experiments have been used to determine their propensity to adopt a PPII helix conformation. Results show that the introduction of three residues of Hyp efficiently stabilizes the PPII helix conformation, while it does not improve the affinity towards the SH3 domain, suggesting that additional forces, e.g., electrostatic interactions, are involved in the SH3_m-cort substrate recognition.     

The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum

Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu(2+), circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein's sequences including 1, N(1)Y(2)G(3)H(4)G(5)G(6)G(7)N(8)Y(9)G(10)N(11), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu(2+) binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO(4) caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu(2+) binding unit.     

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

Background

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.

Results

ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.

Conclusions

We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.     

Secretory production of spider silk proteins in metabolically engineered Corynebacterium glutamicum for spinning into tough fibers

Spider dragline silk is a remarkable fiber made of unique proteins—spidroins—secreted and stored as a concentrated aqueous dope in the major ampullate gland of spiders. This feat has inspired engineering of microbes to secrete spidroins for spinning into tough synthetic fibers, which remains a challenge due to the aggregation-prone feature of the spidroins and low secretory capacity of the expression hosts. Here we report metabolic engineering of Corynebacterium glutamicum to efficiently secrete recombinant spidroins. Using a model spidroin MaSpI16 composed of 16 consensus repeats of the major ampullate spidroin 1 of spider Trichonephila clavipes, we first identified the general Sec protein export pathway for its secretion via N-terminal fusion of a translocation signal peptide. Next we improved the spidroin secretion levels by selection of more suitable signal peptides, multiplexed engineering of the bacterial host, and by high cell density cultivation of the resultant recombinant strains. The high abundance (>65.8%) and titer (554.7 mg L^–1) of MaSpI16 in the culture medium facilitated facile, chromatography-free recovery of the spidroin with a purity of 93.0%. The high solubility of the purified spidroin enabled preparation of highly concentrated aqueous dope (up to 66%) amenable for spinning into synthetic fibers with an appreciable toughness of 70.0 MJ m⁻³. The above metabolic and processing strategies were also found applicable for secretory production of the higher molecular weight spidroin MaSpI64 (64 consensus repeats) to yield similarly tough fibers. These results suggest the good potential of secretory production of protein polymers for sustainable supply of fibrous materials.     

Topological characteristics of helical repeat proteins.

The recent elucidation of protein structures based upon repeating amino acid motifs, including the armadillo motif, the HEAT motif and tetratricopeptide repeats, reveals that they belong to the class of helical repeat proteins. These proteins share the common property of being assembled from tandem repeats of an alpha-helical structural unit, creating extended superhelical structures that are ideally suited to create a protein recognition interface.     

Voltage-dependent anion-selective channels VDAC2 and VDAC3 are abundant proteins in bovine outer dense fibers, a cytoskeletal component of the sperm flagellum

Outer dense fibers (ODF) are specific subcellular components of the sperm flagellum. The functions of ODF have not yet been clearly elucidated. We have investigated the protein composition of purified ODF from bovine spermatozoa and found that one of the most abundant proteins is a 30-32-kDa polypeptide. This protein was analyzed by sequencing peptides derived following limited proteolysis. Peptide sequences were found to match VDAC2 and VDAC3. VDACs (voltage-dependent, anion-selective channels) or eukaryotic porins are a group of proteins first identified in the mitochondrial outer membrane that are able to form hydrophilic pore structures in membranes. In mammals, three VDAC isoforms (VDAC1, -2, -3) have been identified by cDNA cloning and sequencing. Antibodies against synthetic peptides specific for the three mammal VDAC isoforms were generated in rabbits. Their specificity was demonstrated by immunoblotting using recombinant VDAC1, -2, and -3. In protein extracts of bovine spermatozoa, VDAC1, -2, and -3 were detected by specific antibodies, while only VDAC2 and -3 were found as solubilized proteins derived from purified bovine ODFs. Immunofluorescence microscopy of spermatozoa revealed that anti-VDAC2 and anti-VDAC3 antibodies clearly bound to the sperm flagellum, in particular to the ODF. Transmission electron immunomicroscopy supported the finding that VDAC2 protein is abundant in the ODF. Since the ODF does not have any known membranous structure, it is tempting to speculate that VDAC2 and VDAC3 might have an alternative structural organization and different functions in ODF than in mitochondria.     

Conformational analysis of the immunodominant epitopes of the circumsporozoite protein of Plasmodium falciparum and knowlesi

All of the coat proteins of the sporozoite and merozoite stages of Plasmodium, determined to date, contain tandem repeats and most of these contain at least one proline residue. These tandemly repeated segments of the circumsporozite (CS) proteins of P. falciparum and P. knowlesi have been shown to constitute an immunodominant epitope. Antibodies to these peptide segments have been shown to be protective and cause the shedding of the CS protein, known as the CSP reaction. In this study, four synthetic peptides were prepared by solid-phase peptide synthesis. The first peptide corresponds to the tetrapeptide tandem repeat in the CS protein of P. falciparum, repeated eight times, (NANP)₈. The second peptide is an analogue of the first in which glycine is substituted for proline, (NANG)₈. The third peptide corresponds to the tandem repeat of P. knowlesi, PK(1–24), which is repeated twice (QAQGDGANAGQP)₂. The fourth peptide is a tetrapeptide repeat, corresponding to the C-terminal tetrapeptide of PK(1–24) and is repeated eight times, (AGQP)₈. It is shown by CD measurements that the presence of proline in these repeats induces an increase in β-sheet (β-turn) content in the (NANP)₈ peptide relative to the repeat of (NANG)₈ and PK(1–24) peptide in aqueous media. The (AGQP)₈ peptide has the highest β-sheet (β-turn) content in the synthetic peptides. It is concluded that this increase in defined structure correlates well with and hence may contribute to the increased antigenicity in these repeats.     

Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences

BACKGROUND: Mitochondrial processing peptidase (MPP) is a metalloendopeptidase that cleaves the N-terminal signal sequences of nuclear-encoded proteins targeted for transport from the cytosol to the mitochondria. Mitochondrial signal sequences vary in length and sequence, but each is cleaved at a single specific site by MPP. The cleavage sites typically contain an arginine at position -2 (in the N-terminal portion) from the scissile peptide bond in addition to other distal basic residues, and an aromatic residue at position +1. Mitochondrial import machinery recognizes amphiphilic helical conformations in signal sequences. However, it is unclear how MPP specifically recognizes diverse presequence substrates. RESULTS: The crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP complexed with synthetic signal peptides have been determined. MPP is a heterodimer; its alpha and beta subunits are homologous to the core II and core I proteins, respectively, of the ubiquinol-cytochrome c oxidoreductase complex. Crystal structures of two different synthetic substrate peptides cocrystallized with the mutant MPP each show the peptide bound in an extended conformation at the active site. Recognition sites for the arginine at position -2 and the +1 aromatic residue are observed. CONCLUSIONS: MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.     

Visualization of the terminal structure of rice chromosomes 6 and 12 with multicolor FISH to chromosomes and extended DNA fibers

High-resolution fluorescence in situ hybridization (FISH) on interphase and pachytene nuclei, and extended DNA fibers enabled microscopic distinction of DNA sequences less than a few thousands of base pairs apart. We applied this technique to reveal the molecular organization of telomere ends in japonica rice (Oryza sativa ssp. japonica), which consist of the Arabidopsis type TTTAGGG heptameric repeats and the rice specific subtelomeric tandem repeat sequence A (TrsA). Southern hybridizations of DNA digested with Bal31 and EcoRI, and FISH on chromosomes and extended DNA fibers demonstrated that (1) all chromosome ends possess the telomere tandem repeat measuring 3–4 kb; (2) the subtelomeric TrsA occurs only at the ends of the long arms of chromosomes 6 and 12, and measure 6 and 10 kb, which corresponds to 231 and 682 copies for these sites, respectively; (3) the telomere and TrsA repeats are separated by at most a few thousands of intervening nucleotide sequences. The molecular organization for a general telomere organization in plant chromosomes is discussed.     

Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.     

Tandem repeats in proteins: from sequence to structure

The bioinformatics analysis of proteins containing tandem repeats requires special computer programs and databases, since the conventional approaches predominantly developed for globular domains have limited success. Here, I survey bioinformatics tools which have been developed recently for identification and proteome-wide analysis of protein repeats. The last few years have also been marked by an emergence of new 3D structures of these proteins. Appraisal of the known structures and their classification uncovers a straightforward relationship between their architecture and the length of the repetitive units. This relationship and the repetitive character of structural folds suggest rules for better prediction of the 3D structures of such proteins. Furthermore, bioinformatics approaches combined with low resolution structural data, from biophysical techniques, especially, the recently emerged cryo-electron microscopy, lead to reliable prediction of the protein repeat structures and their mode of binding with partners within molecular complexes. This hybrid approach can actively be used for structural and functional annotations of proteomes.