Analyses of the Sequence and Structural Properties Corresponding to Pentapeptide and Large Palindromes in Proteins

The analyses of 3967 representative proteins selected from the Protein Data Bank revealed the presence of 2803 pentapeptide and large palindrome sequences with known secondary structure conformation. These represent 2014 unique palindrome sequences. 60% palindromes are not associated with any regular secondary structure and 28% are in helix conformation, 11% in strand conformation and 1% in the coil conformation. The average solvent accessibility values are in the range between 0–155.28 Ų suggesting that the palindromes in proteins can be either buried, exposed to the solvent or share an intermittent property. The number of residue neighborhood contacts defined by interactions ≤ 3.2 Ǻ is in the range between 0–29 residues. Palindromes of the same length in helix, strand and coil conformation are associated with different amino acid residue preferences at the individual positions. Nearly, 20% palindromes interact with catalytic/active site residues, ligand or metal ions in proteins and may therefore be important for function in the corresponding protein. The average hydrophobicity values for the pentapeptide and large palindromes range between -4.3 to +4.32 and the number of palindromes is almost equally distributed between the negative and positive hydrophobicity values. The palindromes represent 107 different protein families and the hydrolases, transferases, oxidoreductases and lyases contain relatively large number of palindromes.     

Palindromes in proteins

Palindromes in DNA consist of nucleotides sequences that read the same from the 5'-end to the 3'-end, and its double helix is related by twofold axis. They occur in genomes of all organisms and have various functions. For example, restriction enzymes often recognize palindromic sequences of DNA. Palindromes in telomeres are crucial for initiation of replication. One can ask the questions, Do palindromes occur in protein, and if so, what function they play? We have searched the protein SWISSPROT database for palindromic sequences. A great number (26%) of different protein palindromes were found. One example of such protein is systemin, an 18-amino-acid-long peptide. It contains palindrome in its beta-sheet domain that interacts with palindromic fragment of DNA. The other palindrome containing protein is cellular human tumor suppressor p53. Oligonucleotide LTI-ITL has been observed in the crystal structure and is located close to a DNA recognizing domain. As the number of possible palindromic sequences of a given length is far much greater for proteins (20N) than for nucleic acids (4N), the study on their role seems to be an exciting challenge. Our results have clearly showed that palindromes are frequently occurring motives in proteins. Moreover, even very few examples that we have examined so far indicate the importance of further studies on protein palindromes.     

Tyrosine-dependent capture of CAP-Gly domain-containing proteins in complex mixture by EB1 C-terminal peptidic probes

Microtubule dynamics is regulated by an array of microtubule associated proteins of which the microtubule plus-end tracking proteins (+TIPs) are prominent examples. +TIPs form dynamic interaction networks at growing microtubule ends in an EB1-dependent manner. The interaction between the C-terminal domain of EB1 and the CAP-Gly domains of the +TIP CLIP-170 depends on the last tyrosine residue of EB1. In the present study, we generated peptidic probes corresponding to the C-terminal tail of EB1 to affinity-capture binding partners from cell lysates. Using an MS-based approach, we showed that the last 15 amino-acid residues of EB1, either free or immobilized on beads, bound recombinant CAP-Gly domains of CLIP-170. We further demonstrate that this binding was prevented when the C-terminal tyrosine of EB1 was absent in the peptidic probes. Western blotting in combination with a label-free quantitative proteomic analysis revealed that the peptidic probe harboring the C-terminal tyrosine of EB1 effectively pulled-down proteins with CAP-Gly domains from endothelial cell extracts. Additional proteins known to interact directly or indirectly with EB1 and the microtubule cytoskeleton were also identified. Our peptidic probes represent valuable tools to detect changes induced in EB1-dependent +TIP networks by external cues such as growth factors and small molecules.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in D. melanogaster DNA were studied by electron microscopy (EM). The results of these studies differ from the previously published observations regarding the number, distribution and the size of inverted repeats (ir) present in DNA. -1. In contrast to the previous published observation almost all (96%) of the ir were found in crowded clusters. The DNA strands with clustered palindromes contained 2-21 palindromes (4-42 ir), with an average of 7.25 palindromes (14.5 ir) per strand. No correlation could be found between the length of the DNA strands and the number of ir per strand. -2, Also contrary to some previously published results, most (80%) of the ir formed on renaturation unlooped palindromes and these were always clustered. Looped palindromes (hairpins, formed by renaturation of ir separated by a non-homologous sequence long enough to be seen in EM as single-stranded loop) were found 1-2 per DNA strand, as part of clusters or as solitary palindromes in a DNA strand. The average spacing length (inside clusters) between centers of all palindromes was 2.349 kb, and between centers of looped palindromes 7.6 kb. - 3. The length of the ir was found to be smaller than documented in most of the previously published results. The majority, 80-90%, of the ir found in the unlooped and looped palindromes, respectively, belonged to one main-size class with a range of 30-210 bp and an average length of 100 bp, but longer ir were also observed. The average length of the ir in unlooped palindromes was 124 bp, in looped 244 bp, and the total average was 148 bp - 4. It was calculated that there are about 30,000 palindromes (60,000 ir) in the D, melanogaster genome, of which about 24,000 are unlooped and 6,000 looped, with the spacing between centers of all palindromes averaging about 4.4 kb in length.     

Palindromes in Proteins

Palindromes in DNA consist of nucleotides sequences that read the same from the 5′-end to the 3′-end, and its double helix is related by twofold axis. They occur in genomes of all organisms and have various functions. For example, restriction enzymes often recognize palindromic sequences of DNA. Palindromes in telomeres are crucial for initiation of replication. One can ask the questions, Do palindromes occur in protein, and if so, what function they play? We have searched the protein SWISSPROT database for palindromic sequences. A great number (26%) of different protein palindromes were found. One example of such protein is systemin, an 18-amino-acid-long peptide. It contains palindrome in its β-sheet domain that interacts with palindromic fragment of DNA. The other palindrome containing protein is cellular human tumor suppressor p53. Oligonucleotide LTIITL has been observed in the crystal structure and is located close to a DNA recognizing domain. As the number of possible palindromic sequences of a given length is far much greater for proteins (20^N) than for nucleic acids (4^N), the study on their role seems to be an exciting challenge. Our results have clearly showed that palindromes are frequently occurring motives in proteins. Moreover, even very few examples that we have examined so far indicate the importance of further studies on protein palindromes.     

Comparison of physical and genetic properties of palindromic DNA sequences.

Some viable palindromic DNA sequences were found to cause an increase in the recovery of genetic recombinants. Although these palindromes contained no Chi sites, their presence in cis caused apparent recA+-dependent recombination to increase severalfold. This biological property did not correlate with the physical properties of the palindromes' extrusion of cruciform structures in vitro. Thus, two unrelated palindromes with similar effects on recombination in both Escherichia coli and Pseudomonas syringae displayed quite different kinetics of cruciform formation. In plasmids of native superhelical density, one palindrome underwent rapid cruciform formation at 55 degrees C, whereas the other did not form detectable cruciforms at any temperature. A shorter palindrome with similarly rapid kinetics of cruciform formation did not affect recombination detectably. The lack of a clear relationship between physical and genetic properties was also demonstrated in the case of longer, inviable palindromes. Here we found that the degree of asymmetry required in vivo to rescue a long palindrome from inviability far exceeded that required to kinetically prohibit cruciform extrusion in vitro.     

Structure and distribution of inverted repeats (palindromes)

The size and distribution of renatured inverted repeats (palindromes) in Mus musculus DNA were examined by electron microscopy (EM). The majority (85%) of palindromes were found to be clustered in about one half of the DNA strands. The rest of the DNA strands were seen with a solitary looped structure. — The unlooped palindromes constituted 53% of all palindromes and were always clustered. There was a significant reduction in the number of unlooped palindromes in comparison to D. melanogaster DNA (Biezunski, 1981) and as a result the palindrome clusters were smaller and contained 2–8 palindromes [4–16 inverted repeats (ir)] per DNA strand. The looped palindromes had a wide and regular distribution with spacing lengths similar to those found in D. melanogaster DNA, and showed some periodicity. The average spacing between centers of all palindromes (inside a cluster) was 4.325 kb, and between centers of looped palindromes 8.544 kb. — The lengths of the ir of unlooped and looped palindromes were grouped (similar to D. melanogaster DNA) in one size-class with a range of 30–240 bp and an average length of 130 bp. Longer ir were also observed and the average length of ir in unlooped palindromes was 186 bp, in looped 588 bp, and the total average length was 375 bp. — It was calculated that there are about 224,000–320,000 palindromes (ir pairs) in the mouse genome, with the spacing between centers of all palindromes about 13-9 kb in length. — In high molecular weight mouse DNA, complex looped structures composed of rows of 5–8 looped palindromes one on top the other, formed by renaturation of multiple ir, were observed. It is suggested, that clustered repetitive sequences, in direct and inverted orientation, might be of one family and homologous to one another, and be able to reassociate, in vitro and in vivo, into structures of different forms, which could function as binding sites for various regulatory proteins during mouse development.     

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis.     

N-terminal modified cyclopeptidic mimetics of ApolloTBM as inhibitors of TRF2

Telomeric repeat binding factor 2 (TRF2) plays an important role in protecting telomeres from being recognized as DNA breaks. TRF2 performs its telomere protecting functions partially by recruiting a number of accessory proteins to telomeres through its TRF homology (TFRH) domain. Identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions between TRF2 and its associated proteins is crucial for elucidating the molecular mechanisms of these protein–protein interactions. Using a previously identified peptidic mimetic of Apollo_TBM as a lead compound, we designed and synthesized a series of novel TRF2 inhibitors by non-peptidic modifications of the N-terminal residues. These compounds can maintain the binding affinities to TRF2 but have much reduced peptidic characteristics compared to the lead compound.     

Isolation and characterization of DNA-binding proteins from the cyanobacterium Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum) and from spinach chloroplasts

Basic, low-molecular-weight DNA-binding proteins were isolated from the unicellular cyanobacterium Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum) and from the chloroplasts of spinach (Spinacia oleacera). In Synechococcus, two major proteins which bind to double-strand DNA (10 and 16 kDa, respectively) were purified. The 10 kDa protein, named HAq, resembles strongly, in amino-acid composition, eubacterial HU-type proteins. The 16 kDa protein is slightly basic. Its characteristics are compared to those of E. coli protein H1 and 17K. In spinach chloroplasts, a major protein HC (10 kDa), which also binds to ds-DNA, was purified. As observed for known archaebacterial and mitochondrial DNA-binding proteins, its amino-acid composition differs significantly from those of eubacterial HU. The comparison of the amino-terminal sequence (27 residues) with other chloroplast peptidic sequences is discussed.     

Heterodimerization of ecdysone receptor and ultraspiracle on symmetric and asymmetric response elements

Heterodimerization of nuclear receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNA-binding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with ecdysone receptor (EcR) and this complex participates in ecdysone signal transduction. The natural ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using full-length and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to.     

GAP-Seq: a method for identification of DNA palindromes

Background

Closely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes.

Results

We developed a new protocol to identify palindromes that couples the S1 nuclease treated Cot0 DNA (GAPF) with high-throughput sequencing (GAP-Seq). Unlike earlier protocols, it does not involve restriction enzymatic digestion prior to DNA snap-back thereby preserving longer DNA sequences. It also indicates the location of the novel junction, which can then be recovered. Using MCF-7 breast cancer cell line as the proof-of-principle analysis, we have identified 35 palindrome candidates and physically characterized the top 5 candidates and their junctions. Because this protocol eliminates many of the false positives that plague earlier techniques, we have improved palindrome identification.

Conclusions

The GAP-Seq approach underscores the importance of developing new tools for identifying and characterizing palindromes, and provides a new strategy to systematically assess palindromes in genomes. It will be useful for studying human cancers and other diseases associated with palindromes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-394) contains supplementary material, which is available to authorized users.     

Synthesis and physico-chemical properties of peptides in soil humic substances.

Soil humic substances (HS) are heterologous, polydispersive, and multi-functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with 13C-glucose or 15N-nitrate for 8.5 months. Evidence for random-coil peptidic structures in the labeled HS was obtained from 2-D nuclear magnetic resonance (NMR), pyrolysis gas chromatography-mass spectrometry (pyro-GC-MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid-state NMR data. Detailed 13C and 15N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC-MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre-existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.     

α-Helix to β-sheet transition within the LeuiLysj (i = 2j) series of lytic amphipathic peptides by decreasing their size

Summary De novo designed extremely simplified amphipathic basic Leu_iLys_j (i=2j) peptides of 8, 9 and 15 residues were synthesized to clarify the mechanism of action of natural cytotoxic and hemolytic small proteins or peptides. They proved to have strong hemolytic activity towards human erythrocytes which increases with peptide length. These peptides are highly surface active and form stable peptidic films at the air/water interface. The sensitive and efficient FTIR modulated polarization technique (PMIRRAS) allows one to obtain in situ structural and orientational information about the peptides at the interface. A transition of secondary structure is observed: the shorter peptides (8 and 9 residues) adopt β-sheet structures while the longer one (15 residues) is folded into an α-helix. In both cases, the peptides lie with the axis parallel to the interface. Their insertion into a dimyristoylphosphatidylcholine monolayer can be followed from the increase in the surface and/or pressure of the films. In the mixed films, the peptides adopt the same structure and orientation as observed at the air/water interface. Therefore, among the same series of peptides, a transition from β-sheet to α-helix occurs when the length increases (roughly>10 aa), but despite this drastic change both types of structures result in strongly hemolytic peptides.     

The primary structure of stinging nettle (Urtica dioica) agglutinin. A two-domain member of the hevein family.

The primary structure of stinging nettle (Urtica dioica) agglutinin has been determined by sequence analysis of peptides obtained from three overlapping proteolytic digests. The sequence of 80 residues consists of two hevein-like domains with the same spacing of half-cystine residues and several other conserved residues as observed earlier in other proteins with hevein-like domains. The hinge region between the two domains is four residues longer than those between the four domains in cereal lectins like wheat germ agglutinin.     

Viral fusion peptides and identification of membrane-interacting segments

Viral envelope glycoproteins promote infection by mediating fusion between viral and cellular membranes. Fusion occurs after dramatic conformational changes within fusion proteins, leading to the exposure of a short stretch of mostly apolar residues, termed the fusion peptide, which is presumed to insert into the membrane and initiate the fusion process. The typical global composition of fusion peptides, rich in hydrophobic but also in small amino acids such as alanine and glycine, was used here as bait to detect other peptidic segments that can insert into membranes. We so evidenced a similar composition in several cytotoxic peptides, which promote pore formation such as peptides involved in amyloidoses and hydrophobic alpha-hairpins of pore-forming toxins. It is suggested that the structural plasticity observed for several membrane active peptides can be conferred by this particular global amino acid composition, which could be thus used to predict such functional behavior from genome data.