Helical screw sense bias in chiral polyfluorene stimulated by solvent

Conjugated homopolymer poly(9,9‐bis(3‐((S )‐2‐methylbutylpropanoate))fluorene) (PSF) with chiral pendants was synthesized and characterized. Dissolution experiments show that PSF is well dissolved in racemic limonene at high temperature and begins aggregating upon sequential cooling treatment. The corresponding assemblies were transferred to quartz plate by the spin‐coating method. Comparably, film casting from chloroform solution was also prepared. Upon annealing thermal treatments, these PSF films exhibited perfect mirror circular dichroism (CD) Cotton effects and dissymmetry ratios. Optical absorption spectroscopy (UV‐vis), CD, and fluorescence spectroscopy results reveal that chiral side chains successfully induced M ‐ and P ‐helical structures in aggregates and films, and this significant difference was ascribed to their differential supramolecular conformations.     

Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein‐6

Mytilus foot protein type 6 (mfp‐6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp‐6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp‐6 showed high purification yields of 5–6 mg L^?1 cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp‐6.1 protein showed high 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano‐Newton range, we show that rmfp‐6.1 is also able to rescue the oxidation‐dependent adhesion loss of mussel foot protein 3 (mfp‐3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4‐dihydroxyphenyl‐l ‐alanine in mfp‐3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp‐6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587–1593, 2013     

Inhibition of tumor necrosis factor mRNA translation by a rationally designed immunomodulatory peptide

Based on sequences of immunomodulatory peptides derived from the heavy chain of HLA Class I, novel immunomodulatory peptides with increased potency were developed by computer-aided rational design. Allotrap 1258 was characterized in detail and shown to inhibit cell-mediated immune responses in vitro and in vivo. Immunomodulatory activity was associated with the capability of the peptides to modulate heme oxygenase (HO) activity. In this study we analyzed the effect of Allotrap 1258 on cytokine expression. Allotrap 1258 inhibited concanavalin A- and lipopolysaccharide-induced human and mouse tumor necrosis factor (TNF) production in vitro and in vivo but had no effect on interleukin (IL)-1, IL-2, IL-4, IL-6, or IL-10 expression. Experiments with HO-1/KO and iNOS/KO mice showed that Allotrap 1258-mediated inhibition of TNF was independent of HO-1 and iNOS. Quantitation of TNF protein expression and mRNA steady state levels demonstrated that Allotrap 1258-mediated inhibition occurred at the translational level. Deletion of the AU-rich element in the 3'-untranslated region (UTR) of TNF mRNA, a region known to be involved in TNF mRNA translation, had minimal effect on Allotrap 1258-mediated inhibition. However, replacement of the TNF 3'-UTR with the human globin 3'-UTR rendered the peptide inactive. This demonstrates that besides AU-rich elements, other sequences in the 3'-UTR of TNF mRNA are involved in translational control of TNF expression. Such sequences are necessary for Allotrap 1258-mediated inhibition of TNF production.     

Function of the SmpB tail in transfer-messenger RNA translation revealed by a nucleus-encoded form

Stalled bacterial ribosomes are freed when they switch to the translation of transfer-messenger RNA (tmRNA). This process requires the tmRNA-binding and ribosome-binding cofactor SmpB, a beta-barrel protein with a protruding C-terminal tail of unresolved structure. Some plastid genomes encode tmRNA, but smpB genes have only been reported from bacteria. Here we identify smpB in the nuclear genomes of both a diatom and a red alga encoding a signal for import into the plastid, where mature SmpB could activate tmRNA. Diatom SmpB was active for tmRNA translation with bacterial components in vivo and in vitro, although less so than Escherichia coli SmpB. The tail-truncated diatom SmpB, the hypothetical product of a misspliced mRNA, was inactive in vivo. Tail-truncated E. coli SmpB was likewise inactive for tmRNA translation but was still able to bind ribosomes, and its affinity for tmRNA was only slightly diminished. This work suggests that SmpB is a universal cofactor of tmRNA. It also reveals a tail-dependent role for SmpB in tmRNA translation that supersedes a simple role of linking tmRNA to the ribosome, which the SmpB body alone could provide.     

Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids.

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.     

Snake relationships revealed by slow-evolving proteins: a preliminary survey

We present an initial evaluation of relationships among a diverse sample of 215 species of snakes (8% of the world snake fauna) representing nine of the 16 commonly-recognized families. Allelic variation at four slow-evolving. protein-coding loci, detected by starch-gel electrophoresis, was found to be informative for estimating relationships among these species at several levels. The numerous alleles detected at these loci [ Arp -2 (42 alleles). Ltlh -2 (43), Mdh -1 (29), Pgm (Z)] provided unexpected clarity in partitioning these taxa. Most congeneric species and several closely-related genera have the same allele at all four loci or differ at only a single locus. At thc other extreme are those species with three or four unique alleles; these taxa cannot be placed in this analysis. Species sharing two or three distinctive alleles are those most clearly separated into clades. Typhlopids, pythonids, viperids, and elapids were resolved into individual clades. whereas bods were separated into boincs and erycines, and colubrids appeared as scveral distinct clades (colubrines, natricines, psammophines, homalopsines, and xenodontines). Viperids were recognized as a major division containing three separate clades: Asian and American crotalines. Pabearctic and Oriental viperines, and Ethiopian causines. The typhlopids were found to be the basal clade, with the North American erycine boid Chrrrino and the West Indian woodsnakes Tropidophi, Y near the base. A number of species and some small clades were not allocated because of uninformative (common, unique, or conflicting) alleles. Of the 21 S species examined, five to eight appear to have been misplaced in the analysis of these electrophoretic data.     

Direct chiral separation of unnatural amino acids by high-performance liquid chromatography on a ristocetin a-bonded stationary phase.

Direct high-performance liquid chromatographic chiral separation of numerous underivatized unnatural amino acids on a ristocetin A-bonded chiral stationary phase used in the reversed-phase and in the polar organic chromatographic modes is reported. The effects of different parameters such as mobile phase composition, temperature, and the structure of the analytes on the selectivity in both chromatographic modes are discussed. By variation of the parameters, the separation of the stereoisomers was optimized and, as a result, baseline resolution was achieved in most cases.     

Nucleotide bias causes a genomewide bias in the amino acid composition of proteins

We analyzed the nucleotide contents of several completely sequenced genomes, and we show that nucleotide bias can have a dramatic effect on the amino acid composition of the encoded proteins. By surveying the genes in 21 completely sequenced eubacterial and archaeal genomes, along with the entire Saccharomyces cerevisiae genome and two Plasmodium falciparum chromosomes, we show that biased DNA encodes biased proteins on a genomewide scale. The predicted bias affects virtually all genes within the genome, and it could be clearly seen even when we limited the analysis to sets of homologous gene sequences. Parallel patterns of compositional bias were found within the archaea and the eubacteria. We also found a positive correlation between the degree of amino acid bias and the magnitude of protein sequence divergence. We conclude that mutational bias can have a major effect on the molecular evolution of proteins. These results could have important implications for the interpretation of protein-based molecular phylogenies and for the inference of functional protein adaptation from comparative sequence data.     

Pupylated proteins in Corynebacterium glutamicum revealed by MudPIT analysis

In a manner similar to ubiquitin, the prokaryotic ubiquitin‐like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also contain a proteasome. In this study, we set out to study pupylation in the proteasome‐lacking non‐pathogenic model organism Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew aerobically as the parent strain in standard glucose minimal medium, indicating that pupylation is dispensable under these conditions. After expression of a Pup derivative carrying an aminoterminal polyhistidine tag in the Δpup mutant and Ni²⁺‐chelate affinity chromatography, pupylated proteins were isolated. Multidimensional protein identification technology (MudPIT) and MALDI‐TOF‐MS/MS of the elution fraction unraveled 55 proteins being pupylated in C. glutamicum and 66 pupylation sites. Similar to mycobacteria, the majority of pupylated proteins are involved in metabolism or translation. Our results define the first pupylome of an actinobacterial species lacking a proteasome, confirming that other fates besides proteasomal degradation are possible for pupylated proteins.     

Hydrogen-bond disruption probability in proteins by a modified self-consistent harmonic approach

Modified self-consistent harmonic approach was employed to calculate the probability for the disruption of each individual hydrogen bonds (H bonds) in x-ray crystal structure of several proteins. The computed probability for 82% of intraprotein and water-protein H bonds studied were found to be roughly consistent with estimated free energies from protein engineering and hydrogen exchange experiments. Hydrogen bonds have been proposed as part of a stereochemical code for protein folding. Proteins fold into unique three-dimensional structures; therefore those bonds involved in the folding code are expected to be stable. We have applied this method to tens of hydrogen bonds in a protein assumed to be involved in the folding code of a protein. 58% of these H bonds were found to have a lower disruption probability (-1.8 kcal/mol). Our results showed that modified self-consistent harmonic approach might be explored as a method supplement to existing methods in analysis of hydrogen bonds in proteins.     

Novel third-letter bias in Escherichia coli codons revealed by rigorous treatment of coding constraints

A novel bias in codon third-letter usage was found in Escherichia coli genes with low fractions of "optimal codons", by comparing intact sequences with control random sequences. Third-letter usage has been found to be biased according to preference in codon usage and to doublet preference from the following first letter. The present study examines third-letter usage in the context of the nucleotide sequence when these preferences are considered. In order to exclude any influence by these factors, the random sequences were generated such that the amino acid sequence, codon usage, and the doublet frequency in each gene were all preserved. Comparison of intact sequences with these randomly generated sequences reveals that third letters of codons show a strong preference for the purine/pyrimidine pattern of the next codons: purine (R) is preferred to pyrimidine (Y) at the third site when followed by an R-Y-R codon, and pyrimidine is preferred when followed by an R-R-Y, an R-Y-Y or a Y-R-Y codon. This bias is probably related to interactions of tRNA molecules in the ribosome.     

Involvement of post-translational modification of neuronal plasticity-related proteins in hyperalgesia revealed by a proteomic analysis

To clarify roles of an endogenous pain modulatory system of the central nervous system (CNS) in hyperalgesia, we tried to identify qualitative and quantitative protein changes by a proteomic analysis using an animal model of hyperalgesia. Specifically, we first induced functional hyperalgesia on male Wistar rats by repeated cold stress (specific alternation of rhythm in temperature, SART). We then compared proteomes of multiple regions of CNS and the dorsal root ganglion between the hyperalgetic rats and non-treated ones by 2-D PAGE in the pI range of 4.0-7.0. We found that SART changed the proteomes prominently in the mesencephalon and cerebellum. We thus analyzed the two brain regions in more detail using gels with narrower pI ranges. As a result, 29 and 23 protein spots were significantly changed in the mesencephalon and the cerebellum, respectively. We successfully identified 12 protein spots by a MALDI-TOF/TOF MS and subsequent protein database searching. They included unc-18 protein homolog 67K, collapsin response mediator protein (CRMP)-2 and CRMP-4, which were reported to be involved in neurotransmitter release or axon elongation. Interestingly, mRNA expression levels of these three proteins were not changed significantly by the induction of hyperalgesia. Instead, we found that the detected changes in the protein spots are caused by the post-translational modification (PTM) of proteolysis or phosphorylation. Taken together, development of the hyperalgesia would be linked to PTM of these three CNS proteins. PTM regulation may be one of the useful ways to treat hyperalgesia.     

Codon bias as a factor in regulating expression via translation rate in the human genome

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.     

Nuclei of HeLa cells interactomes unravel a network of ghost proteins involved in proteins translation

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Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.     

Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree

We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.