Backbone 1H, 15N and 13C resonance assignments of the 27kDa fluorescent protein mCherry.

mCherry is one of the most successfully applied monomeric red fluorescent proteins (RFPs) for in vivo and in vitro imaging. However, questions pertaining to the photostability of the RFPs remain and rational further engineering of their photostability requires information about the fluorescence quenching mechanism in solution. To this end, NMR spectroscopic investigations might be helpful, and we present the near-complete backbone NMR chemical shift assignment to aid in this pursuit.

Structural and Functional Characterization of the Newly Designed Antimicrobial Peptide Crabrolin21.

(1) Background: antimicrobial resistance is becoming a dramatic problem for public health, and the design of new antimicrobial agents is an active research area. (2) Methods: based on our previous work, we designed an improved version of the crabrolin peptide and characterized its functional and structural properties with a wide range of techniques. (3) Results: the newly designed peptide, crabrolin21, is much more active than the previous ones and shows specific selectivity towards bacterial cells. (4) Conclusions: crabrolin21 shows interesting properties and deserves further studies.

Impact of Anion Exchange Ionomers on the Electrocatalytic Performance for the Oxygen Reduction Reaction of B-N Co-doped Carbon Quantum Dots on Activated Carbon.

Composite electrocatalytic electrodes made from B-N co-doped carbon quantum dots (CQD) and various anion exchange ionomers (AEI) are studied for the oxygen reduction reaction (ORR) in alkaline solutions. The quantity and positions of dopants in CQD, prepared by hydrothermal synthesis, are analyzed by various spectroscopies, including 11B NMR spectroscopy that evidenced boronic acid at edge sites. The AEI are synthesized with various backbones, including more hydrophilic polysulfone, hydrophobic poly(alkylene biphenyl), and poly(2,6-dimethyl-1,4-phenylene oxide) with intermediate hydrophilicity; the functional groups are trimethylammonium moieties grafted on long (LC) or short (SC) side chains. The CQD/AEI ink is drop-casted on activated carbon paper, and the samples are fixed on a rotating disk electrode and studied in three-electrode configuration in oxygen-saturated 0.1 M KOH. The onset potentials are among the best in the literature (Eonset ≈ 0.94 V vs RHE). The highest electrocatalytic activity is observed for electrodes containing AEI with long side chains; the sample containing PPO LC attains excellent ORR currents approaching that of benchmark Pt/C cloth. The electrocatalytic performances are discussed in view of the many relevant AEI parameters, including hydrophilicity, oxygen permeability, catalyst dispersivity, and ionic conductivity.

Solution structure of the anticancer p28 peptide in biomimetic medium.

The p28 peptide derived from Pseudomonas aeruginosa azurin shows an anticancer activity after binding to p53 protein and is currently in Phase I of clinical trials. We have studied its structure in water and in a biomimetic media and show that the peptide is unstructured in water but when studied in a biomimetic medium assumes a structure very similar to the one observed in azurin, suggesting a high propensity of this peptide to maintain this secondary structure. Analysis of p28 sequences from different bacterial species indicates conservation of the secondary structure despite amino acid replacement in different positions, suggesting that others, similar peptides could be tested for binding to p53.

A Strategy Based on Loop Analysis to Develop Peptide Epitopes: Application to SARS-CoV-2 Spike Protein.

Many current strategies for inducing an immune response rely on the production of an antigenic protein. Such methods can be problematic if the folding of the antigenic protein is incorrect. To avoid this problem, we propose a method based on grafting specific regions of the chosen antigenic protein onto biocompatible polymeric matrices, so that they can mimic portions of the antigenic protein. These regions are selected following the criterion according to which they are not folded, are exposed to the solvent and are not already present in the human body, so that they are not recognized by the immune system as self. Regions are selected using the primary sequence of the protein and, where possible, its tertiary structure. The application of this strategy to the Spike protein of SARS-CoV-2 is presented.

Nanocomposite Anion Exchange Membranes with a Conductive Semi-Interpenetrating Silica Network.

Nanocomposite anion exchange membranes were synthesized based on poly(sulfone trimethylammonium) chloride. A hybrid semi-interpenetrating silica network containing a large amount of quaternary ammonium groups was prepared by two sol-gel routes, in situ with a single precursor, N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TMSP), or ex situ mixing two precursors, TMSP and 3-(2-aminoethylamino)propyldimethoxy-methylsilane (AEAPS). The properties of these hybrid composites and their degradation after immersion in 1 M KOH at 60 °C were studied. The degradation is reduced in the composite materials with a lower decrease in the ion exchange capacity. FTIR spectra showed that a main degradation mechanism with a single precursor TMSP is the dissolution of the hybrid silica network in KOH, whereas it is stable with the mixture of TMSP/AEASP. This conclusion is in agreement with the thermogravimetric analysis. The mechanical properties show a better ductility with a single precursor and higher stiffness and strength, but less ductility, by the ex situ route. The activation energy was between 0.25 and 0.14 eV for Cl and OH ion conduction, respectively, consistent with the migration mechanism.

Effect of positive charges in the structural interaction of crabrolin isoforms with lipopolysaccharide.

Antimicrobial peptides (AMPs) appear as chemical compounds of increasing interest for their role in killing bacteria and, more recently, for their ability to bind endotoxin (lipopolysaccharide, LPS) that is released during bacterial infection and that may lead to septic shock. This dual role in the mechanism of action can further be enhanced in a synergistic way when two or more AMPs are combined together. Not all AMPs are able to bind LPS, suggesting that several modes of binding to the bacterial surface may exist. Here we analyze a natural AMP, crabrolin, and two mutated forms, one with increased positive charge (Crabrolin Plus) and the other with null charge (Crabrolin Minus), and compare their binding abilities to LPS. While Crabrolin WT as well Crabrolin Minus do not show binding to LPS, the mutated Crabrolin Plus exhibits binding and forms a well defined structure in the presence of LPS. The results strengthen the importance of positive charges for the binding to LPS and suggest the mutated form with increased positive charge as a promising candidate for antimicrobial and antiseptic activity.

Crabrolin, a natural antimicrobial peptide: structural properties.

A joint application of experimental and computational approaches has revealed the exceptionally high attitude of crabrolin, a 13-residue peptide with sequence FLPLILRKIVTAL-NH2 , to adopt alpha-helix conformation not only in membrane-mimicking solvents but also in the presence of a not negligible amount of water. Our study shows that this propensity essentially resides in the intrinsic thermodynamic stability of alpha-helix conformation whose kinetic stability is drastically reduced in water solvent. Our analysis suggests that this is due to two effects enhanced by water: a more local effect consisting of the demolition of intra-peptide H-bonds, essential for the alpha-helix formation, and a bulk - electrostatic - effect favoring conformational states more polar than alpha-helix. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Evidence for a quadruplex structure in the polymorphic hs1.2 enhancer of the immunoglobulin heavy chain 3' regulatory regions and its conservation in mammals.

Regulatory regions in the genome can act through a variety of mechanisms that range from the occurrence of histone modifications to the presence of protein-binding loci for self-annealing sequences. The final result is often the induction of a conformational change of the DNA double helix, which alters the accessibility of a region to transcription factors and consequently gene expression. A ∼300 kb regulatory region on chromosome 14 at the 3' end (3'RR) of immunoglobulin (Ig) heavy-chain genes shows very peculiar features, conserved in mammals, including enhancers and transcription factor binding sites. In primates, the 3'RR is present in two copies, both having a central enhancer named hs1.2. We previously demonstrated the association between different hs1.2 alleles and Ig plasma levels in immunopathology. Here, we present the analysis of a putative G-quadruplex structure (tetraplex) consensus site embedded in a variable number tandem repeat (one to four copies) of hs1.2 that is a distinctive element among the enhancer alleles, and an investigation of its three-dimensional structure using bioinformatics and spectroscopic approaches. We suggest that both the role of the enhancer and the alternative effect of the hs1.2 alleles may be achieved through their peculiar three-dimensional-conformational rearrangement. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 768-778, 2016.

Time-resolved assembly of a nucleoprotein complex between Shigella flexneri virF promoter and its transcriptional repressor H-NS.

The virF gene of Shigella, responsible for triggering the virulence cascade in this pathogenic bacterium, is transcriptionally repressed by the nucleoid-associated protein H-NS. The primary binding sites of H-NS within the promoter region of virF have been detected here by footprinting experiments in the presence of H-NS or its monomeric DNA-binding domain (H-NSctd), which displays the same specificity as intact H-NS. Of the 14 short DNA fragments identified, 10 overlap sequences similar to the H-NS binding motif. The 'fast', 'intermediate' and 'slow' H-NS binding events leading to the formation of the nucleoprotein complex responsible for transcription repression have been determined by time-resolved hydroxyl radical footprinting experiments in the presence of full-length H-NS. We demonstrate that this process is completed in ≤1 s and H-NS protections occur simultaneously on site I and site II of the virF promoter. Furthermore, all 'fast' protections have been identified in regions containing predicted H-NS binding motifs, in agreement with the hypothesis that H-NS nucleoprotein complex assembles from a few nucleation sites containing high-affinity binding sequences. Finally, data are presented showing that the 22-bp fragment corresponding to one of the HNS binding sites deviates from canonical B-DNA structure at three TpA steps.

Phenyl derivative of iron 5,10,15-tritolylcorrole.

The phenyl-iron complex of 5,10,15-tritolylcorrole was prepared by reaction of the starting chloro-iron complex with phenylmagnesium bromide in dichloromethane. The organometallic complex was fully characterized by a combination of spectroscopic methods, X-ray crystallography, and density functional theory (DFT) calculations. All of these techniques support the description of the electronic structure of this phenyl-iron derivative as a low-spin iron(IV) coordinated to a closed-shell corrolate trianion and to a phenyl monoanion. Complete assignments of the (1)H and (13)C NMR spectra of the phenyl-iron derivative and the starting chloro-iron complex were performed on the basis of the NMR spectra of the regioselectively ß-substituted bromo derivatives and the DFT calculations.

Quantitative HSQC Analyses of Lignin: A Practical Comparison.

Lignin is the second-most abundant polymer after cellulose within the biomass of our planet. Structurally, it displays random oligomeric units without fixed repetition schemes beyond the stage of dimers. Quantitative (1)H-(13)C HSQC measurements have recently greatly facilitated lignin analyses. In some cases, however, long acquisition times needed for obtaining quantitative HSQCs are not compatible with the chemical integrity of (a potentially functionalised) lignin sample. We thus compared different methods that were developed for more time-efficient quantitative HSQC measurements with respect to their usefulness in lignin analyses: reliable and reproducible results were obtained using both the QQ-HSQC and the HSQC0 method.

Structural and functional characterization of the bacterial translocation inhibitor GE82832.

The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome- and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit "head" which accompanies the ribosome-unlocking step of translocation.

Milled wood lignin: a linear oligomer.

The degree of polymerization (DP) of softwood and hardwood milled wood lignin samples and their branching degrees were quantitatively evaluated by a novel end-group titration approach composed of QQ-HSQC, (31)P NMR, and DFRC coupled with (31)P NMR analysis techniques. The DP of lignin can be calculated when the C9 formula, the amounts of phenolic groups, pinoresinol (ß-ß), diphenylethane (ß-1), and phenolic diphenyl (5-5') lignin subunits have been determined. Data on the degree of polymerization of lignin obtained by NMR techniques were not affected by supramolecular aggregation processes. (31)P NMR analysis coupled with DFRC and QQ-HSQC allowed a detailed evaluation of the occurrence of condensed units in lignin and showed the terminal nature of diphenyl ether and diphenyl subunits. The resulting data unequivocally show that milled wood lignin is a linear oligomer.

Elucidation of lignin structure by quantitative 2D NMR.

Quick quantitative HSQC (QQ-HSQC) was applied to quantitative evaluation of different inter-unit linkages in an array of milled softwood and hardwood and technical lignins by using the guaiacyl C2 and syringyl C2-C6 signals as internal standards. The results were found to be highly reproducible and comparable with earlier literature reports. The advantage of QQ-HSQC NMR analysis of lignin is contemporary detection and quantification of lignin inter-unit linkages with a direct, non-destructive method requiring short acquisition times.

Sequence-specific recognition of DNA by the C-terminal domain of nucleoid-associated protein H-NS.

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage lambda CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::lambdaCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::lambdaCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr(109), Arg(113), Thr(114), and Ala(116)) belonging to the same protein loop and Glu(101). The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.

The solution structure of the monomeric copper, zinc superoxide dismutase from Salmonella enterica: structural insights to understand the evolution toward the dimeric structure.

The structure of the SodCII-encoded monomeric Cu, Zn superoxide dismutase from Salmonella enterica has been solved by NMR spectroscopy. This represents the first solution structure of a natural and fully active monomeric superoxide dismutase in solution, providing information useful for the interpretation of the evolutional development of these enzymes. The protein scaffold consists of the characteristic beta-barrel common to the whole enzyme family. The general shape of the protein is quite similar to that of Escherichia coli Cu, Zn superoxide dismutase, although some differences are observed mainly in the active site. SodCII presents a more rigid conformation with respect to the engineered monomeric mutants of the human Cu, Zn superoxide dismutase, even though significant disorder is still present in the loops shaping the active site. The analysis of both dynamics and hydration properties of the protein in solution highlights the factors required to maintain the fully active and, at the same time, monomeric protein. This study provides novel insights into the functional differences between monomeric and dimeric bacterial Cu, Zn superoxide dismutases, in turn helping to explain the convergent evolution toward a dimeric structure in prokaryotic and eukaryotic enzymes of this class.

NMR assignment of reduced form of copper, zinc superoxide dismutase from Salmonella enterica.

Almost complete assignment (97%) of NMR resonances was obtained for the reduced, Cu(I), form of prokaryotic CuZnSOD from Salmonella enterica. 13C direct detection was used to complement the standard bouquet of 1H detected triple resonance experiments and contributed to the identification of proline backbone resonances and to side chains assignments of Asx, Glx and aromatic rings. This is the only complete assignment available for monomer SOD from prokaryotic organisms.

Purified promyelocytic leukemia coiled-coil aggregates as a tetramer displaying low alpha-helical content.

The promyelocytic leukemia (PML) gene is involved in the 15/17 chromosomal translocation of acute promyelocytic leukemia (APL). It encodes a nuclear phosphoprotein containing an alpha-helical coiled-coil domain with four heptad repeats. The heptad repeats consist of four clusters of hydrophobic amino acids that mediate in vivo the complex formation between PML and other PML molecules or PML-RARalpha mutant protein. In this report, we show the production of PML coiled-coil (fragment 223-360) as a fusion protein, its solubilization by the combined action of two different detergents, and its purification with affinity chromatography after column proteolytic cleavage. The FPLC chromatograms of the purified coiled-coils, carried out under non-denaturing conditions, show that the peptide elutes only in the presence of Sarkosyl detergent (conc. 0.1%) and, under these conditions, elutes as a tetrameric complex. This confirms the evidence from in vivo experiments that this region is responsible for protein complex formation. The HPLC analyses show the presence of a single peak eluting under highly hydrophobic conditions, indicating the high hydrophobicity of the peptide in accordance with the primary sequence analysis. Finally, the purified peptide was structurally characterized by means of circular dichroism (CD) measurements that were carried out with low Sarkosyl concentration (0.003%). The CD spectra indicate a low alpha-helical content (13.5%) with respect to predictions based on the primary sequence analysis (PSI-PRED, SS-PRO, and J-PRED), suggesting that the alpha-helix content could be modulated by coiled-coil surrounding domains and/or by other post-translational modifications, even if the effect of the Sarkosyl on the peptide secondary structure cannot be excluded.